EP2971132A1 - Biomarqueurs de miarn à base de tissu et de sang pour le diagnostic, le pronostic et le potentiel prédictif de métastases dans le cancer colorectal - Google Patents
Biomarqueurs de miarn à base de tissu et de sang pour le diagnostic, le pronostic et le potentiel prédictif de métastases dans le cancer colorectalInfo
- Publication number
- EP2971132A1 EP2971132A1 EP14764952.9A EP14764952A EP2971132A1 EP 2971132 A1 EP2971132 A1 EP 2971132A1 EP 14764952 A EP14764952 A EP 14764952A EP 2971132 A1 EP2971132 A1 EP 2971132A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- mir
- expression
- level
- mirna
- patient
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
- C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
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Definitions
- the present invention relates generally to the fields of oncology, molecular biology, cell biology, and cancer. More particularly, it concerns cancer diagnosis, prognosis or classification using molecular markers.
- CRC Colorectal cancer
- CEA levels do not always correlate with the presence of metastasis, and the incidence of false-positive and false-negative results are very high (Fakih 2006; Tan 2009). Consequently, there is a dire need to identify highly robust biomarkers that can clinically determine cancer prognosis, and are better indicators of patient outcome than the existing TNM staging system or other conventional tumor markers of CRC (Duffy 2001).
- MicroRNAs are non-coding RNA molecules of approximately 21-23 nucleotides in length that regulate target gene expression by interfering with their transcription or by inhibiting translation (Cortez 2009). miRNAs play crucial roles in diverse cellular biological processes, including differentiation, proliferation, growth, migration and survival. The discovery that miRNA expression is frequently dysregulated in malignant tumors underpins their critical role, which is a matter of active investigation, both from a basic science perspective and for its clinical usefulness (van Kouwenhove 2011).
- biomarkers may be used for the detection or assessment of pathologies, including, but not limited to all stages of cancer, such as colorectal cancer.
- Certain embodiments may comprise methods for evaluating a colorectal cancer such as a primary colorectal cancer in a patient suspected of having or determined to have a colorectal cancer such as primary colorectal cancer, or providing a prognosis or diagnosis for the patient.
- the methods may provide a clinician with information useful for screening, examination, surveillance, diagnosis and/or treatment options.
- Methods may involve identifying a patient suspected of having or determined to have a colorectal cancer such as primary colorectal cancer.
- the methods may further include determining or measuring in a sample from the patient expression levels of one, two, three, four, five, six, seven, eight, nine, or more biomarker miRNA such as miR-21, miR-31, miR124, miR-200c, miR-203, miR-885-5p, let-7i, miR-lOb, miR-320a, and/or miR221.
- the measuring may also include measuring methylation levels of one or more genes encoding biomarker miRNA such as miR124, miR-200c and/or miR-203.
- the methods may comprise measuring expression levels of miR-885-5p as the single miRNA biomarker or in combination with one of more miRNA biomarkers described herein.
- the sample may be a blood sample or serum sample.
- the sample may be a tissue sample.
- the miR-885-5p marker may be measured in a blood sample or serum sample or any sample from a circulation system.
- methods may involve measuring the expression of one or more markers, such as miR-21, miR-31, miR-200c, miR-203 and/or miR-885-5p in a serum sample or any sample from a circulation system.
- methods may also include measuring the expression of one or more markers, such as miR-124, let-7i, miR-lOb, miR-320a, and/or miR-221 or the methylation of a gene encoding miR-124, miR-200c, and/or miR-203 in a tissue sample.
- the methods may comprise determining a risk associated with colorectal cancer in the patient based on the level of expression compared to a control or reference level for the biomarker.
- the risk may be a risk for dysplasia, cancer or metastasis, or more particularly, distant metastasis.
- the risk may be a risk of having or developing cancer, of having a poor prognosis, of having poor survival probability, or developing tumor recurrence or metastasis, or a combination thereof.
- the risk may be a risk of developing liver metastasis.
- the risk may be a risk of developing distant metastasis.
- monitoring may comprise sensitive detection technologies used to measure minimal residual disease (MRD), recurrence and resistance for managing or treating cancer patients.
- MRD minimal residual disease
- the methods may comprise determining the patient as having a high or significant risk, such as a poor diagnosis or prognosis or a high metastasis risk by having increased expression level of miR-885-5p or increased expression levels in one or more of miR-21, miR- 31, miR-124, miR-200c, miR-203, miR-885-5p, miR-lOb, and/or miR-221 or deceased expression levels of miR-124, let-7i, and 320a compared to a control or reference expression level, or an increased methylation level of a gene encoding miR-124 compared to a control or reference methylation level.
- a high or significant risk such as a poor diagnosis or prognosis or a high metastasis risk by having increased expression level of miR-885-5p or increased expression levels in one or more of miR-21, miR- 31, miR-124, miR-200c, miR-203, miR-885-5p, miR-lOb, and/or mi
- the method may comprise determining the patient as having a low risk, such as a favorable diagnosis or prognosis or a low metastasis risk by having decreased expression levels in a biomarker comprising miR-21, miR-31, miR-124, miR-200c, miR-203, miR-885-5p, miR-lOb, and/or miR-221 or increased expression levels of miR-124, let-7i, and 320a compared to a control or reference expression level.
- a biomarker comprising miR-21, miR-31, miR-124, miR-200c, miR-203, miR-885-5p, miR-lOb, and/or miR-221 or increased expression levels of miR-124, let-7i, and 320a compared to a control or reference expression level.
- the expression or methylation levels of the biomarker in the sample may be compared to a control or reference levels for the biomarker.
- the increased or decreased expression or methylation n levels with respect to reference levels or control may be indicative of a high risk of colorectal dysplasia, cancer, or metastasis.
- the control may be a normal tissue, a non-cancerous tissue, a pre-cancer tissue, a primary tumor tissue, a non- dysplastic tissue, a non-metastasized tissue, or the same tissue taken at a point in time before the patient develops dysplasia, cancer or any stages of cancer, or metastasis or from patients with poor or favorable prognosis or diagnosis.
- the reference level can be expression or methylation levels of any of the controls or an average of a population of controls or expression or methylation levels of a different gene or miRNA taken from the same tissue or a different tissue whose expression or methylation level does not change, for example in developing dysplasia, cancer, metastasis.
- methods involve comparing the level of expression or methylation of at least one biomarker miRNA to the level of expression or methylation level of a comparative miRNA to determine a biomarker difference value.
- a "comparative miRNA” refers to a miRNA whose expression level is used to evaluate the level of another miRNA in the sample; in some embodiments, the expression level of a comparative miRNA is used to evaluate a biomarker miRNA expression level.
- a level of miRNA is increased or decreased compared to a control or reference level if it is at least 20, 30, 40, 50, 60., 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, or 1000% higher (or any range derivable therein) than the reference or control level.
- This may or may not include using a standardized or normalized level of expression in determining whether there is an increase or decrease.
- the level may be an expression level of the miRNA or methylation level of the encoding gene.
- Embodiments also concern methods and compositions that can be used for detecting colorectal cancer, differentiating colorectal cancer, distinguishing colorectal cancer, colorectal cancer as a high risk lesion, identifying colorectal cancer as a low/high risk metastasis, identifying tissue having colorectal cancer as a target for surgical resection or intensive or frequent surveillance, determining tissue having colorectal cancer that should not be surgically resected, categorizing colorectal cancer, diagnosing colorectal cancer, providing a prognosis to a patient regarding colorectal cancer, evaluating management, surveillance or treatment options for colorectal cancer, or treating a patient with colorectal cancer.
- methods involve measuring or determining in a sample from the patient that the sample has increased expression levels in a biomarker comprising miR-21, miR-31, miR-200c, miR-203, miR-885-5p, miR-lOb, and/or miR-221 or deceased expression levels of miR-124, let-7i, and 320a compared to a control or reference expression level for the gene and identifying the patient as being at a significant risk for developing colon dysplasia, cancer, or metastasis compared to the overall risk for normal people or patients without colorectal dysplasia or cancer or patients with colorectal dysplasia or cancer.
- methods involve managing a patient suspected of having or determined to have a colorectal cancer such as primary colorectal cancer by measuring the expression of miR-21, miR-31, miR124, miR-200c, miR-203, miR-885-5p, let-7i, miR-lOb, miR-320a, and/or miR221 in a sample, and monitoring and/or treating the patient for colorectal cancer or metastasis if the patient exhibits one or more characterizes including: increased expression level of miR-885-5p or increased expression levels in one or more of miR-21, miR- 31, miR-124, miR-200c, miR-203, miR-885-5p, miR-lOb, and/or miR-221 or deceased expression levels of miR-124, let-7i, and 320a compared to a control or reference expression level, or an increased methylation level of a gene encoding miR-124 compared to a control or reference
- the methods may comprise identifying the patient as being suspected of having or determined to have a colorectal cancer and having one or more of the following: increased expression level of miR-885-5p or increased expression levels in one or more of miR-21, miR-31, miR-124, miR-200c, miR-203, miR-885-5p, miR-lOb, and/or miR-221 or deceased expression levels of miR-124, let-7i, and 320a compared to a control or reference expression level, or an increased methylation level of a gene encoding miR-124 compared to a control or reference methylation level.
- the methods may further comprise developing a management plan or administering a preventive procedure, screening, further diagnosis, examination, monitoring, surveillance plan or treatment to the identified patient based on the different levels.
- the methods may comprise administering a preventive procedure or treatment that inhibits or reduces the expression levels of one or more markers of miR-21, miR-31, miR-124, miR-200c, miR-203, miR-885-5p, miR-lOb, and/or miR- 221 and/or a preventive procedure or treatment that increases or promotes the expression levels of one or more markers of miR-124, let-7i, and 320a.
- methods and compositions for isolating, enriching, and/or labeling miRNA molecules and for preparing and using arrays or other detection techniques for miRNA analysis may refer to U.S.7,919,245 (incorporated herein by reference).
- methods involve obtaining a sample of a subject or a patient or obtaining a sample from the subject or patient.
- subject or patient may refer to an animal (for example a mammal), including but not limited to humans, non-human primates, rodents, dogs, or pigs.
- the methods of obtaining provided herein include methods of biopsy such as fine needle aspiration, core needle biopsy, vacuum assisted biopsy, incisional biopsy, excisional biopsy, punch biopsy, shave biopsy or skin biopsy.
- methods involve obtaining a serum sample or tissue sample.
- the tissue sample may be a rectal, cecum, or colon tissue sample or any sample of a large intestine.
- the sample is obtained from a biopsy from rectal, cecum, or colon tissue by any of the biopsy methods previously mentioned.
- the sample may be obtained from any of the tissues provided herein that include but are not limited to gall bladder, skin, heart, lung, breast, pancreas, liver, muscle, kidney, smooth muscle, bladder, intestine, brain, prostate, esophagus, or thyroid tissue.
- the sample may include but not be limited to blood, serum, sweat, hair follicle, buccal tissue, tears, menses, urine, feces, or saliva.
- the sample may be a tissue sample, a whole blood sample, a urine sample, a saliva sample, a serum sample, a plasma sample or a fecal sample.
- the sample is obtained from cystic fluid or fluid derived from a tumor or neoplasm.
- the cyst, tumor or neoplasm is in the digestive system.
- any medical professional such as a doctor, nurse or medical technician may obtain a biological sample for testing.
- the patient or subject may obtain a biological sample for testing without the assistance of a medical professional, such as obtaining a whole blood sample, a urine sample, a fecal sample, a buccal sample, or a saliva sample.
- the sample may be a fresh, frozen or preserved sample or a fine needle aspirate.
- the sample is a formalin-fixed, paraffin- embedded (FFPE) sample.
- An acquired sample may be placed in short term or long term storage by placing in a suitable medium, excipient, solution, or container. In certain cases storage may require keeping the sample in a refrigerated, or frozen environment. The sample may be quickly frozen prior to storage in a frozen environment. In certain instances the frozen sample may be contacted with a suitable cryopreservation medium or compound.
- cryopreservation mediums or compounds include but are not limited to: glycerol, ethylene glycol, sucrose, or glucose.
- Some embodiments further involve isolating nucleic acids such as ribonucleic or
- R A from a biological sample.
- Other steps may or may not include amplifying a nucleic acid in a sample and/or hybridizing one or more probes to an amplified or non-amplified nucleic acid.
- a microarray may be used to measure or assay the level of miRNA expression in a sample.
- Measuring or assaying for expression levels of an miRNA can be accomplished by a variety of different chemical and/or enzymatic reactions that are well known to those of skill in the art.
- methods may involve, but not be limited to, next generation sequencing, single-molecule real-time sequencing, mass spectrometry, digital color-coded barcode technology analysis, microarray expression profiling, quantitative PCR, reverse transcriptase PCR, reverse transcriptase real-time PCR, quantitative real-time PCR, end-point PCR, multiplex end-point PCR, cold PCR, ice-cold PCR, in situ hybridization, Northern hybridization, hybridization protection assay (HP A), branched DNA (bDNA) assay, rolling circle amplification (RCA), single molecule hybridization detection, invader assay, and/or Bridge Litigation Assay.
- next generation sequencing single-molecule real-time sequencing, mass spectrometry, digital color-coded barcode technology analysis, microarray expression profiling, quantitative PCR, reverse transcriptase PCR
- Non-limiting amplification methods may include real-time PCR (quantitative PCR (q- PCR)), digital PCR, nucleic acid sequence-base amplification (NASBA), ligase chain reaction, multiplex ligatable probe amplification, invader technology (Third Wave), rolling circle amplification, in vitro transcription (IVT), strand displacement amplification, transcription- mediated amplification (TMA), RNA (Eberwine) amplification, and other methods that are known to persons skilled in the art.
- Measuring or assaying for methylation levels of a miRNA can be accomplished by a variety of different chemical and/or enzymatic reactions that are well known to those of skill in the art.
- Non-limiting methylation detection methods may include next generation sequencing, single-molecule real-time sequencing, nanopore sequencing; mass spectrometry, bisulfite sequencing, combined bisulfite restriction analysis (COBRA), Southern blotting, single nucleotide primer extension (SNuPE), methylation-specific PCR (MSPCR), restriction landmark genomic scanning for methylation (RLGS-M), Hpall-tmy fragment enrichment by ligation- mediated PCR (HELP assay), CpG island microarray, ChlP-chip (chromatin immnuprecipitation-on-chip), ChlP-seq (chromatin immunoprecipitation- sequencing), methylated DNA immunoprecipitation (MeDIP), or a microarray-based methylation profiling.
- next generation sequencing single-molecule real-time sequencing,
- Methods may further involve recording the expression levels, risk, diagnosis, or prognosis in a tangible medium, reporting it to the patient, a health care payer, a physician, an insurance agent, or an electronic system, monitoring the patient for colorectal dysplasia, cancer, or metastasis, and/or comprising determining or administering a further screening, examination, monitoring, surveillance, or treatment for the patient based on the expression levels, risk, diagnosis or prognosis within one hour, one day, one week, one month, one year, two years, three years, four years, five years of the measuring or evaluating or within any intermediate time values or ranges.
- Some further embodiments involve normal surveillance for colorectal dysplasia, cancer, or metastasis or administering a colorectal dysplasia, cancer, or metastasis prevention or treatment if the patient does not have a high risk based on expression levels.
- the treatment may comprise inhibiting or reducing the expression levels of the biomarkers or any traditional cancer therapies, such as surgery, chemotherapy, radiation, gene therapy, or immunotherapy for patients with risks determined based on the biomarker expression levels.
- Further embodiments involve methods of managing a patient suspected of having or determined to have a colorectal cancer in a patient.
- the methods may comprise monitoring by performing colonoscopy or other testing methods, or treating the patient for colorectal cancer or metastasis after the patient has been determined to have increased expression of miR-885-5p or any miRNA different levels described herein, or a combination thereof.
- cyst or tumor or biopsy may first undergo cytological examination or evaluation prior to implementing any molecular tests.
- Another embodiment provides methods for evaluating a subject suspected of having or determined to have a colorectal cancer, comprising (a) providing a tissue sample or biological fluid obtained from said subject; (b) determining the level of expression of at least one micro ribonucleic acid (miRNA) in said sample from the subject; and (c) comparing the level of expression determined in step (b) to a control or reference level of the same miRNA.
- said at least one of the miRNA determined by this method is selected from the group consisting of let-7i, miR-lOb, miR-21, miR-31, miR-124, miR-200c, miR-203, miR-221, miR-320a, and miR-885-5p.
- step (b) comprises determining the expression level of at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 of said miRNAs.
- the control level represents the expected level found in a population of normal (cancer-free) subjects, and/or the reference level of miRNA is the expression level in a non-cancerous tissue of the same subject.
- the method may further comprise determining the level of a normalizing nucleic acid.
- the biological fluid is selected from the group consisting of blood, serum and plasma.
- the miRNA is selected from the group consisting of miR- 21, miR-31, miR-200c, miR-203, and miR-885-5p.
- miRNA level is determined in a tissue sample taken from a tumor found in the subject, which may be a primary tumor.
- the level of expression of let-7i found in a primary tumor is lower compared to the control level found in a population of normal (cancer-free) subjects, and/or the level of miR-lOb found in a primary tumor is elevated compared to the control level found in a population of normal (cancer-free) subjects, the subject is further examined for metastatic tumors.
- the expression level of miRNA is determined in a biological fluid selected from blood, serum and plasma, and the reference level is determined in a tissue sample of a primary tumor in the same subject, for prognosis of the subject. In particular, differences between the expression levels in these two biological samples may be prognostic, especially if the miRNA is miR-21, miR-31, miR-203, miR-200c, or miR-885.
- Evaluation according to this invention may be combined with other diagnostic or prognostic procedures; for example, endoscopic examination of the subject may be contingent on whether the level of expression of miRNA determined in step (b) is elevated relative to the control or reference level.
- the method may further comprise endoscopic removal of polyps and/or adenomas from the subject.
- an elevated level of miRNA is found in a biological fluid, especially if the elevated miRNA is from the group consisting of miR-21, miR-31, miR-200c, miR-203, and miR-885-5p, the subject is further examined for metastatic tumors.
- the reference level is determined in the same tissue sample or biological fluid obtained from the same subject at an earlier time.
- the subject has been treated for colorectal cancer, and additional treatment is conditioned on the comparison between the level of expression of the at least one miRNA before treatment to the level of expression of the same miRNA after treatment.
- methods will involve determining or calculating a diagnostic or risk score based on data concerning the expression level of one or more miRNAs, meaning that the expression level of the one or more miRNAs is at least one of the factors on which the score is based.
- a diagnostic score will provide information about the biological sample, such as the general probability that the patient is at high or significant risk for developing dysplasia or cancer, or is at low risk for developing dysplasia or cancer, or both.
- the diagnostic score represents the probability that the patient is more likely than not either at high or low risk for dysplasia, cancer or metastasis.
- a probability value is expressed as a numerical integer or number that represents a probability of 0% likelihood to 100% likelihood that a patient has a particular category of cancer, dysplasia, metastasis, or risk, such as high risk or low risk for dysplasia, cancer or metastasis.
- the probability value is used to predict a patient that is at risk for development of metastasis or a patient that is at risk for development of colon cancer.
- the probability value is expressed as a number that represents a probability of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% likelihood (or any range derivable therein) that a patient has a particular category of cancer, dysplasia, met
- a difference between or among weighted coefficients or expression levels or between or among the weighted comparisons may be, be at least or be at most about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 15.5, 16.0, 16.5, 17.0, 17.5, 18.0, 18.5, 19.0, 19.5, 20.0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48
- determination of calculation of a diagnostic, prognostic, or risk score is performed by applying classification algorithms based on the expression values of biomarkers with differential expression p values of about, between about, or at most about 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.011, 0.012, 0.013, 0.014, 0.015, 0.016, 0.017, 0.018, 0.019, 0.020, 0.021, 0.022, 0.023, 0.024, 0.025, 0.026, 0.027, 0.028, 0.029, 0.03, 0.031, 0.032, 0.033, 0.034, 0.035, 0.036, 0.037, 0.038, 0.039, 0.040, 0.041, 0.042, 0.043, 0.044, 0.045, 0.046, 0.047, 0.048, 0.049, 0.050, 0.051, 0.052, 0.053, 0.054, 0.055, 0.056,
- any of the methods described herein may be implemented on tangible computer- readable medium comprising computer-readable code that, when executed by a computer, causes the computer to perform one or more operations.
- a tangible computer-readable medium comprising computer-readable code that, when executed by a computer, causes the computer to perform operations comprising: receiving information corresponding to the level of expression of a first biomarker comprising miR-885-5p in a serum sample of a patient suspected of having or determined to have a colorectal cancer such as primary colorectal cancer; and determining a difference value in the expression level using the information corresponding to the expression level in the serum sample compared to a control or reference level.
- receiving information comprises receiving from a tangible data storage device information corresponding to the expression or methylation levels from a tangible storage device.
- the medium further comprises computer- readable code that, when executed by a computer, causes the computer to perform one or more additional operations comprising: sending information corresponding to the difference value to a tangible data storage device, calculating a risk score for the patient of developing dysplasia or cancer or metastasis, developing a management performing intensive or frequent surveillance to the patient for colorectal dysplasia or cancer or metastasis or administering a dysplasia or cancer or metastasis prevention or treatment if the patient has a high risk, and/or or performing normal surveillance for colorectal dysplasia or cancer or metastasis or administering a less aggressive or conventional prevention or treatment for dysplasia or cancer or metastasis if the patient does not have a high risk.
- kits containing the disclosed compositions or compositions used to implement the disclosed methods can be used to evaluate one or more miR A molecules.
- a kit contains, contains at least, or contains at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more, or any range and combination derivable therein, miRNA probes or primers including those that may specifically hybridize under stringent conditions to miRNAs disclosed herein.
- kits or methods may involve 1, 2, or more miRNA probes or primers, which may be capable of specifically detecting any biomarkers for methylation or expression.
- the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.
- A, B, C, or combinations thereof refers to all permutations and combinations of the listed items preceding the term.
- A, B, C, or combinations thereof is intended to include at least one of: A, B, C, AB, AC, BC, or ABC, and if order is impairment in a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB.
- expressly included are combinations that contain repeats of one or more item or term, such as BB, AAA, MB, BBC, AAABCCCC, CBBAAA, CABABB, and so forth.
- the skilled artisan will understand that typically there is no limit on the number of items or terms in any combination, unless otherwise apparent from the context.
- compositions and/or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.
- FIG. 1 shows the expression analysis of metastasis predictive microRNAs expression comparing Primary Colorectal (PC) cancer microRNA expression compared to colorectal cancer (CRC) liver metastasis (LM) microRNA expression.
- PC Primary Colorectal
- CRC colorectal cancer
- LM liver metastasis
- FIG. 2 Figure 2 is an analysis of the expression of the miR-200 family (-200b, -
- FIG. 3 ⁇ shows the results of the qRT-PCR validation for selected miRNAs in 58
- FIG. 4 shows the results from the microarray validation for selected miRNAs in 84 PCs.
- FIG. 5 ⁇ shows the results of qRT-PCR validation for miR-7i (left graph) and miR-lOb (right graph) in 175 PCs.
- FIG. 6 shows the ISH validation for the expression of miR-7i and miR-lOb in
- FIG. 7 Expression status of candidate miRNAs as CRC metastasis-specific biomarkers.
- 4 were down-regulated (let-7i, miR-320a, miR-lOb and miR-221) and 2 were up- regulated (miR-30b and miR-885-5p) in liver metastasis (LM) compared to primary CRC (pCRC) in an independent validation cohort of 59 pairs of matching pCRC and LM tissues.
- LM liver metastasis
- pCRC primary CRC
- FIGS. 8A-8B In situ expression of CRC metastasis-specific miRNAs (let-7i, miR-lOb, and miR-885-5p.
- Pathologic expression patterns of three validated CRC metastasis- specific miRNAs were determined by hybridization with LNA-modified and 5'- and 3'-DIG-labeled oligonucleotide probes.
- pCRC positive and negative controls
- B matched primary pCRC with and without later metastases
- FIGS. 9A-9B Kaplan-Meier Overall Survival Analysis based on 4 miRNAs
- FIGS. 10A-10B Kaplan-Meier Overall Survival Analysis based on 2 miRNAs
- FIGS. 11A-11B Kaplan-Meier Overall Survival Analysis based on Tissue
- FIG. 12 Study Process Flow Chart
- FIGS. 13A-13H Expression of miR-21 and miR-31 in culture media of CRC cell lines (HCT116 and SW620).
- MiR-21 levels in media of both HCT116 (A) and SW620 (B) increased with increased cell counts (0.5 - 2 x 10 6 cells/well) and longer incubation intervals, while MiR-31 levels did not change in either cell line (C, D).
- Y-axis represents relative expression of miR-21 and miR-31 normalized to cel-miR-39.
- Y-axis (loglO-scale) represents relative expression of miR-21 normalized to cel-miR-39. Boxes represent the interquartile range and the horizontal line across each box indicates median values. Statistically significant differences were determined using the Mann-Whitney U test and Kruskal-Wallis tests. *P ⁇ 0.05; *** O.0001. Receiver operating characteristics (ROC) curve analysis using serum miR-21 for distinguishing patients with colorectal neoplasms from normal controls.
- ROC Receiver operating characteristics
- Serum miR-21 yielded an AUC value of 0.927 (95% CI: 0.89-0.96), with 82.8%) sensitivity and 90.6%> specificity in distinguishing CRC from nomlal controls.
- C Serum miR-21 yielded AUC values of 0.803 (95% CI: 0.71-0.88) with 76.8% sensitivity and 81.1% specificity in discriminating adenomas from normal controls.
- AUC values derived from ROCCH curves of original and BCa bootstrap-corrected samples were 0.935 (95%> CI: 0.812- 0.982) and 0.919 (95% CI: 0.867-0.958), respectively in distinguishing CRC patients from normal controls.
- E AUC values derived from ROCCH curves of original and BCa bootstrap- corrected samples were 0.838 (95% CI: 0.619-0.964) and 0.813 (95% CI: 0.691-0.910), respectively in distinguishing adenoma patients from normal controls.
- FIGS. 15A-15B Validation of miR-21 expression in matched tissue samples
- (n 186).
- Y-axis (loglO scale) represents relative expression of miR-21 normalized to miR-16 in tissue samples. Boxes represent the interquartile range and the horizontal line across each box indicates median value. Statistically significant differences were determined using the Mann- Whitney U test and Kruskal- Wallis tests. *, ⁇ 0.05: ⁇ 0.0001.
- FIGS. 16A-16C Alterations in serum miR-21 expression levels in patients with
- B Comparison of serum miR-21 levels in 45 CRC patients who underwent potentially curative surgeries.
- C Comparison of serum miR-21 levels in 15 CRC patients with non-curative surgeries.
- Y-axis (log 10 scale) represents relative expression of miR-21 normalized to cel-miR-39. Boxes represent the interquartile range and the horizontal line across each box indicates the median value. Statistically significant differences were determined using the Wilcoxon test.
- FIGS. 17A-17B Kaplan-Meier survival analysis in CRC patients based upon miR-21 expression in primary tumors and mate/zed serum samples.
- FIGS. 19A-19D Validation of miR-200c expression in a validation cohort of
- FIGS. 20A-20B Kaplan-Meier survival curves of CRC patients subdivided by miR-200c levels in serum and matched primary tumors from CRC patients.
- the miRNA expression cut-off thresholds for miR-200c expression in serum were deduced from the ROC curves with Youden's Index.
- FIGS 21A-21D Expression of miR-200c in tissues from CRC patients.
- B Dot plots of miR-200c tissue levels across various CRC stages.
- D In situ hybridization analysis of miR-200c in matched primary CRCs and their corresponding LM. Representative photomicrographs are shown from 2 primary CRCs (left panels) and matched LM (right panels). MiR-200c expressions in LMs were higher than that in primary CRCs expressed.
- miR-200c expression in normal liver tissues was either very low or absent. Line indicates the mean value. Expression levels of miR-200c were normalized to has-miR-16. Statistically significant differences were determined using Mann- Whitney tests and Kruskal-Wallis test.
- FIGS. 22A-22B Serum miR-200c expression based upon N stage in curative patients.
- A Dot plots of serum miR-200c levels based upon N Stage in stage I-III CRC patients.
- B Dot plots of serum miR-200c levels subdivided by N Stage in stage II+III CRC patients. The lines indicate the mean values. Expression levels of miR-200c (loglO scale on the y-axis) were normalized to cel-miR-39. Statistically significant differences were determined using Mann- Whitney tests and Kruskal-Wallis test.
- FIGS. 23A-23B Serum miR-200c expression and Kaplan-Meier survival curves of CRC patients who underwent curative surgery.
- FIGS. 25A-25D Receiver operating characteristics (ROC) curve analysis using serum miR-21 for distinguishing patients with colorectal neoplasms from normal controls.
- A miR-124 methylation levels yielded an AUC value of 0.960 (95% CI: 0.92-0.98), with 89.1% sensitivity and 90.0% in distinguishing CRC from healthy mucosa
- B AUC value of 0.942 for discriminating CRC from adjacent normal mucosa from CRC patients, with 88.0% sensitivity and 97.4% specificity.
- C miR-124 methylation levels yielded an AUC value of 0.977 (95% CI: 0.91-0.99), with 96.5% sensitivity and 90.0% specificity in distinguishing adenoma (AD) from healthy mucosa (NN)
- D AUC value of 0.985 (95% CI: 0.96-0.99) for discriminating adenoma (AD) from adjacent normal mucosa (NC) from CRC patients, with 91.2%) sensitivity and 99.4% specificity.
- FIGS. 26A-26F Correlation between miR-124 methylation and Age in healthy colonic mucosa (NN), adjacent normal mucosa (NC), adenoma (AD) and colorectal cancer (CRC)
- C, D No correlation between miR-124 methylation and Age in adenoma and CRC.
- FIGS. 27A-27B Kaplan-Meier survival analysis in CRC patients based upon miR-124 methylation and expression in primary CRC samples.
- FIGS. 28A-28C Functional analysis in vitro after transfection of either miR-
- A MTT assay showed that restoration of miR-124 inhibits cell growth in both Lovo and HCT-116 cell lines at 24h, 48h and 72h after transfection.
- B Invasion assay shows Transfection of miR-124 precursor inhibits invasion ability of Lovo and HCT-116 cells compared to negative control precursor.
- C Wound healing assay shows that transfection of miR-124 precursor inhibits migration ability of both cell lines compared to negative control precursor.
- FIGS. 29A-29C Strategy for identification of potential targets of miR-124 in
- CRC CRC.
- A The Venn diagram represents the down-regulated genes (>1.5 fold change) observed in the gene microarray analysis after transfection of miR-124 precursor (in blue), and the predicted targets with seeding sequences of miR-124 was selected by in silico prediction tool miRecords (in red).
- B The list of genes identified using mentioned strategy. The expression intensity of each mRNA.
- C Comparison between microarray analysis and RT-PCR. For each MAPRE1 and PPM IF gene, the variation in expression compared to control is represented as average fold change for microarray and comparison of expression levels between miR-124 precursor transfected cells and control cells.
- FIGS. 30A-30E Small-scale examinations of miR-200 family and miR-203 expression in serum with Stage I and Stage IV CRC patients. Box plots of serum levels of miR- 200b (A), miR-200c (B), miR-203 (C), miR-429 (D) and miR-141 (E) in Stage I and Stage IV from 12 CRC patients, respectively. MiR-200c and miR-203 levels in serum with stage IV are significantly higher than that in stage I CRC patients. The box represents the interquartile range and the line across the box indicates the median value. Expression levels of miR-200c and miR- 203 (log 10 scale on the y-axis) are normalized to cel-miR-39. Statistically analysis was performed using Mann-Whiteny tests.
- B Box plot of serum miR-200c level across TNM stage.
- D Box plot of serum miR-203 level across TNM stage.
- MiR-200c and miR-203 levels in serum with CRC are significantly elevated compared to those in normal control and each miRNA level in stage IV CRC patients is significantly higher than that in stage I-II.
- the Box represents the interquartile range and the line across the box indicates the median value.
- Expression levels of miR-200c and miR-203 are normalized to cel- miR-39. Statistically significant differences were determined using Mann-Whiteny tests and Kruska-Wallis test. The patient number of stage I, II, III and IV is 44, 59, 41 and 40, respectively.
- FIGS. 32A-32D Box plots of serum miR-200c and miR-203 level in patients with CRC subdivided by Clinical H stage and Pathological N stage.
- FIGS. 33A-33B Kaplan-Meier survival curves subdivided by miR-200c and miR-203 levels in serum from CRC patients.
- B The overall survival rate of CRC patients with high miR-203 expression in serum was significantly lower than that of those with low miR-203 expression (p ⁇ 0.0001; Log-rank test).
- FIGS. 34A-34F Large-scale validation of miR-200c and miR-203 in primary tumor samples from 92 out of 184 CRC patients.
- A Box plot of tissue miR-200c level across TNM stage.
- B Box plot of tissue miR-200c level across in comparison between stage I-III and stage IV CRC patients.
- C Box plot of tissue miR-203 level across TNM stage.
- D Box plot of tissue miR-203 level across in comparison between stage I-III and stage IV CRC patients.
- Figure 35A-35E shows expression status of miR-203 in human clinical specimens and CRC cell culture medium.
- A Expression status of miR-203 in 58 pairs primary CRC (pCRC) and their corresponding matching liver metastasis (LM). (The gray horizontal bar represents mean expression levels; *** O.001, t test)
- B In situ hybridization analysis of miR- 203 in pCRC and corresponding LMs. (Positive control, U6 snRNA; Negative control, scrambled miRNA control)
- C Released amount of miR-203 in the HT-29 culture medium was increased depend on cell number and culture duration.
- TNM Tumor Node Metastasis
- Figure 36A-36E shows Kaplan-Meier survival analysis for miR-203 expression in serum and matched tissue specimens of CRC patients, and miR-203 expression status in different classification of lymph node (N classification), liver (H classification), and peritoneal (P classification) metastases.
- N classification lymph node
- H classification liver
- P classification peritoneal
- C lymph node (N classification; NO, absent lymph node metastasis; Nl, metastasis in 1 to 3 regional lymph node; N2, metastasis in 4 or more regional lymph node; N3, lymph node metastasis to the aorta),
- D liver (H classification; HO, no liver metastasis; HI, representing liver metastasis with less than 5 nodules smaller than 5 cm; H2, indicative of metastasis that does not involve HI and H3; H3, indicating liver metastasis with more than 5 metastasis larger than 5 cm)
- E peritoneal (P classification; P0, no peritoneal metastasis; PI, metastasis localized to the adjacent peritoneum; P2, limited metastasis to the distant peritoneum; P3, diffuse metastasis to the distant peritoneum) metastasis.
- Figure 37A-37D shows results for an in vivo liver metastasis mouse model.
- Athymic nude mice were sacrificed 10 weeks after injection of HT-29 CRC cells (2.0 X 10 6 /50 of PBS) in the spleen.
- A Representative photo of non-treated control mouse.
- B Representative photo of liver metastasis established mouse. Yellow color arrows indicate metastatic foci.
- the upper-right panel shows the representative result of H&E staining of the mice liver.
- the lower-left panel displays the representative result of ISH staining for miR-203 of the mice liver.
- Embodiments of the present invention involve biomarkers and methods for detecting Colorectal Cancer (CRC) metastasis and exploring curative target of metastasized CRC, including but not limited to cancer research, cancer screening, diagnosis of metastasis, planning of cancer treatment and molecular target of anti-cancer drug.
- CRC Colorectal Cancer
- Certain aspects of the present invention include the identification and use of miRNA biomarkers (let-7i, miR-lOb, miR-30b, miR-21, miR-31, miR-34a, miR-141, miR-200b, miR-200c, miR-203, miR-221, miR-320a, miR-373, miR-429, miR-518d, and miR-520c) that have been found to be very specific for detecting liver metastasized CRC. Most of the existing cancer metastasis biomarkers are developed through comparison between primary tissues with metastasis and without metastasis.
- certain aspects of the present invention may be based on a detailed analysis and discovery that certain miRNA biomarkers were identified by direct comparison between primary CRC and matching liver metastasis tissues, rather than a comparison to primary tissues. These biomarkers were validated using tissue sample miRNAs expression, but also using serum samples of CRC patients with distant metastasis. Thus, the miRNA biomarkers in certain aspects of the present invention are more accurate and specific compared to biomarkers developed using just primary cancer tissues.
- the miRNAs biomarkers may be used as metastasis specific biomarkers because they are derived from the direct comparison between primary CRC and matching liver metastasis tissues.
- the miRNAs biomarkers are more specific for the detection of CRC metastasis, as validated using miRNAs expression of serum samples from CRC patients with and without distant metastasis.
- Diagnosis may refer to the process of attempting to determine or identify a possible disease or disorder, or to the opinion reached by this process. From the point of view of statistics the diagnostic procedure may involve classification tests.
- Prognosis may refer to a prediction of how a patient will progress, and whether there is a chance of recovery.
- Cancer prognosis generally refers to a forecast or prediction of the probable course or outcome of the cancer.
- cancer prognosis includes the forecast or prediction of any one or more of the following: duration of survival of a patient susceptible to or diagnosed with a cancer, duration of recurrence-free survival, duration of progression free survival of a patient susceptible to or diagnosed with a cancer, response rate in a group of patients susceptible to or diagnosed with a cancer, duration of response in a patient or a group of patients susceptible to or diagnosed with a cancer, and/or likelihood of metastasis in a patient susceptible to or diagnosed with a cancer.
- Prognosis may also include prediction of favorable responses to cancer treatments, such as a conventional cancer therapy.
- subject or “patient” is meant any single subject for which therapy is desired, including humans, cattle, dogs, guinea pigs, rabbits, chickens, and so on. Also intended to be included as a subject are any subjects involved in clinical research trials not showing any clinical sign of disease, or subjects involved in epidemiological studies, or subjects used as controls.
- colonal cancer includes the well-accepted medical definition that defines colorectal cancer as a medical condition characterized by cancer of cells of the intestinal tract below the small intestine (i.e., any portion of the large intestine (colon), including the cecum, ascending colon, transverse colon, descending colon, sigmoid colon, or rectum). Additionally, as used herein, the term “colorectal cancer” also further includes medical conditions, which are characterized by cancer of cells of the duodenum and small intestine (jejunum and ileum).
- tissue sample includes any material composed of one or more cells, either individual or in complex with any matrix obtained from a patient.
- the definition includes any biological or organic material and any cellular subportion, product or by-product thereof.
- tissue sample should be understood to include without limitation colorectal tissue samples, tissues suspected of including colorectal cancer cells, blood components, and even fecal matter or fluids that includes colorectal cells.
- tissue for purposes of this invention are certain defined acellular structures such as dermal layers of epithelium that have a cellular origin but are no longer characterized as cellular.
- biological fluid refers to a fluid containing cells and compounds of biological origin, and may include blood, stool or feces, lymph, urine, serum, pus, saliva, seminal fluid, tears, urine, bladder washings, colon washings, sputum or fluids from the respiratory, alimentary, circulatory, or other body systems.
- biological fluids the nucleic acids containing the biomarkers may be present in a circulating cell or may be present in cell-free circulating DNA or R A.
- the term "gene” refers to a functional protein, polypeptide or peptide-encoding unit. As will be understood by those in the art, this functional term includes both genomic sequences, cDNA sequences, or fragments or combinations thereof, as well as gene products, including those that may have been altered by the hand of man. Purified genes, nucleic acids, protein and the like are used to refer to these entities when identified and separated from at least one contaminating nucleic acid or protein with which it is ordinarily associated.
- allele or “allelic form” refers to an alternative version of a gene encoding the same functional protein but containing differences in nucleotide sequence relative to another version of the same gene.
- nucleic acid or “nucleic acid molecule” refers to polynucleotides, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), oligonucleotides, fragments generated by the polymerase chain reaction (PCR), and fragments generated by any of ligation, scission, endonuclease action, and exonuclease action.
- DNA deoxyribonucleic acid
- RNA ribonucleic acid
- PCR polymerase chain reaction
- Nucleic acid molecules can be composed of monomers that are naturally-occurring nucleotides (such as DNA and RNA), or analogs of naturally-occurring nucleotides (e.g., a-enantiomeric forms of naturally-occurring nucleotides), or a combination of both.
- Modified nucleotides can have alterations in sugar moieties and/or in pyrimidine or purine base moieties.
- Sugar modifications include, for example, replacement of one or more hydroxyl groups with halogens, alkyl groups, amines, and azido groups, or sugars can be functionalized as ethers or esters.
- the entire sugar moiety can be replaced with sterically and electronically similar structures, such as aza-sugars and carbocyclic sugar analogs.
- modifications in a base moiety include alkylated purines and pyrimidines, acylated purines or pyrimidines, or other well-known heterocyclic substitutes.
- Nucleic acid monomers can be linked by phosphodiester bonds or analogs of such linkages. Analogs of phosphodiester linkages include phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoranilidate, phosphoramidate, and the like.
- nucleic acid molecule also includes so-called “peptide nucleic acids,” which comprise naturally-occurring or modified nucleic acid bases attached to a polyamide backbone. Nucleic acids can be either single stranded or double stranded.
- a “biomarker” refers to a molecular indicator that is associated with a particular pathological or physiological state.
- the "biomarker” as used herein is a molecular indicator for cancer, more specifically an indicator for primary CRCs and distant metastasis of primary CRCs.
- Examples of “biomarkers” include miR-885-5p, let-7i, miR-lOb, miR-21, miR-31, miR-30b, miR-34a, miR-124, miR-141, miR-200b, miR-200c, miR-203, miR- 221, miR-320a, miR-373, miR-429, miR-518d, and miR-520c.
- biomarker miRNA refers to a miRNA whose expression level is indicative of a particular disease or condition.
- the level of expression of a biomarker miRNA may highlight or emphasize differences in miRNA expression between different populations, such as low or high risk cancer, particularly colorectal cancer. In some embodiments, when miRNA expression is different in a particular population relative to another population, differences between miRNA expression levels can be increased, decreased, highlighted, emphasized, or otherwise more readily observed.
- kit denotes combinations of reagents and adjuvants required for an analysis.
- test kit consists in most cases of several units, one-piece analysis elements are also available, which must likewise be regarded as testing kits.
- TNM refers to the internationally recognized TNM classification of malignant tumors developed and maintained by the International Union against Cancer, which has been adopted by the American Joint Committee on Cancer (AJCC) and the International Federation of Gynecology and Obstetrics (FIGO).
- AJCC American Joint Committee on Cancer
- FIGO International Federation of Gynecology and Obstetrics
- Biomarker microRNAs are short RNA molecules (e.g., 16-29 nucleotides in length) that arise from longer precursors, which are transcribed from non-protein coding genes (Carrington el al, 2003). The precursors are processed by cellular proteins to generate short double-stranded miRNA. One of the miRNA strands is incorporated into a complex of proteins and miRNA called the RNA-induced silencing complex (RISC). The miRNA guides the RISC complex to a target mRNA, which is then cleaved or translationally silenced, depending on the degree of sequence complementarity of the miRNA or its target mRNA (Bagga et al, 2005; Lim et al, 2005).
- RISC RNA-induced silencing complex
- miRNAs are differentially expressed in cancers, including CRC (Calin 2006; Schetter 2008; Lu 2005; Balaguer 2010; Balaguer 2011; Link 2010).
- MiRNA signatures are emerging as promising biomarkers for diagnosis, prognosis and metastasis for cancer patients (Schetter 2008; Bloomston 2007).
- Most studies to date have focused on the role for miRNAs in primary tumors but not the metastatic foci.
- a more systematic and comprehensive analysis is necessary to determine the specific involvement of miRNAs in CRC metastasis.
- miRNAs are relatively stable and measurable, making them ideal targets for molecular diagnostics.
- the miR-885-5p form of this microRNA acts as a tumour suppressor in neuroblastoma, through interference with cell cycle progression and cell survival. It is found at 3p25.3, a chromosome region frequently deleted in primary neuroblastoma, and expression results in p53 protein accumulation and pathway activation. Altered expression of multiple genes is observed with miR-885-5p, including the CDK2 and MCM5 genes encoding cyclin- dependent kinase 2 and mini-chromosome maintenance protein MCM5, and also with several p53 target genes.
- serum miR-885-5p may be used as a non-invasive CRC biomarker.
- MiR-885-5p was significantly up-regulated in LM compared to pCRC tissues, and high serum miR-885-5p expression was an independent factor for predicting CRC prognosis and metastasis, suggesting that the elevated levels of serum miR-885-5p may be due to up-regulation of this miRNA in distant, metastasized cells.
- let7i and miR-lOb were significantly down-regulated in LM compared to pCRC.
- Low let-7i expression and high miR-lOb expression were independent predictors of distant metastasis in pCRC tissues. Since each miRNA can regulate hundreds of different target genes, 6 ' 7 aberrantly expressed miRNAs in cancer tissues reflect the possible mechanistic basis of genetic dysregulation in multiple signaling pathways.
- miRNAs may be used as both tissue and/or serum-based CRC metastasis biomarkers may be clinically applicable for the management of patients with CRC.
- EMT Epithelial-to-mesenchymal transition
- This contributes to accelerated invasiveness, dissemination and metastasis of epithelial tumor cells in several carcinomas, including CRC (Spaderna 2006; Hugo 2007; Thiery 2002).
- the miR-200 family inhibits the E-cadherin-suppressor targets such as zinc finger E-box binding homeobox-1 (ZEB1) and 02 (ZEB2), which are important initiators of EMT in CRC (Burk 2008; Wellner 2009).
- MiR-200c/429 cluster was found to be significantly over-expressed in liver metastasis compared to primary colorectal cancer, and the expression of these miRNAs was specifically regulated by aberrant expression of their promoter regions (Hur 2012). In spite of their involvement in metastasis, none of the previous studies has explored the clinical significance of miR-200 family expression in serum of patients with CRC.
- serum levels of miR-200c which is a bona fide EMT-related miRNA, are not only significantly associated with a metastatic phenotype in the colon, but also serve as a potential biomarker for predicting lymph node metastasis, tumor recurrence and prognosis in CRC patients.
- MiR-21 is an "oncogenic miRNA" that modulates the expression of multiple cancer-related target genes such as PTEN, TPM1 and PDCD, has been shown to be overexpressed in various human tumors (Meng 2007; Zhu 2007; Asangani 2008).
- miR-21 expression is up-regulated in CRC tissues, is elevated during tumor progression, and is also associated with poor survival and response to chemotherapy (Slaby 2007; Shibuya 2010; Chang 2011; Schetter 2008). The clinical significance of circulating miR-21 levels in CRC remains unclear at this time.
- miR-21 is another miRNA frequently overexpressed in CRC tissues and has been shown to associate with tumor prognosis (Slaby 2007; Wang 2009). Additionally, both miR-21 and miR-31 are significantly upregulated even in premalignant lesions, such as colonic adenomas, which are the target lesions of CRC screening (Oberg 2011; Fassan 2011; Cekaite 2012). It is envisaged that these two miRNAs might be good candidates for exploration as circulating biomarkers for the early detection and prognosis of CRC, assuming that the expression pattern for these miRNAs in serum mirrors that in the neoplastic tissues.
- miR-21 levels in cell culture medium increased significantly in a time- and cell number- dependent manner, establishing the secretory nature of this miRNA for development as a noninvasive biomarker of the early detection of colorectal neoplasia.
- Another interesting feature was the existence of significant correlation between miR-21 expression in primary lesions and those in sera.
- the fact that the circulating miR-21 in serum of CRC patients is likely produced by the CRCs was further strengthened by an observation that a significant drop in serum miR-21 expression in post-operative serum vis-a-vis pre-operative samples following potentially curative surgery in patients with CRC.
- miR-21 expression may be exploited not only as a promising non-invasive biomarker for early detection of CRC, but also for the identification of clinically meaningful adenomas - a critical target lesion for any CRC screening strategy.
- miR-21 expression in serum may also serve as a prognostic biomarker for CRC.
- Examples described herein clearly highlight that the relative expression for serum miR-21 was significantly different between cases (patients with adenomas and cancers) and controls. Unlike tissue or cellular miRNAs, at present, due to the lack of availability of a consensus house-keeping miRNA for normalizing the expression of circulating miRNAs (Cortez 2011; Zen 2012), measurement of "relative expression levels" of circulating miRNAs has been the common approach in published studies.
- MiR-203 has been shown to directly suppress EMT activators such as ZEB2 and
- the present inventors have examined the clinical significance of miR-203 expression in CRC metastasis using pairs of pCRC and corresponding matching LM tissues as well as serum and matched tissue specimens from CRC patients. Their results indicate that high serum miR-203 expression is significantly associated with poor survival and metastasis progression in CRC patients. Moreover, these studies provide the first evidence that circulating miR-203 in serum originates from distant metastasized cancer cells through direct analysis of miR-203 expression in primary CRC tissues and matching metastatic CRC tissues present in the liver.
- miR-203 expression is significantly up-regulated in liver metastases compared to matching primary CRC tissues.
- serum miR-203 expression was positively associated with progression of liver metastasis, but matching primary CRC tissue miR-203 expression was not.
- ISH analysis clearly revealed the specific high expression of miR-203 in CRC cells that had metastasized to the liver. Taken together, this evidence collectively indicates that high levels of serum miR-203 expression may be due to up- regulation of its expression in distant CRC cells that have undergone the process of metastasis.
- miR-203 expression analyses in matching pCRC and serum cohorts herein suggest that tissue miR-203 is not associated with clinicopathological factors of CRC patients, whereas serum expression of miR-203 has significant clinical importance in cancer cell dissemination to lymph node, distant organs, liver, and peritoneal cavity as well as patient survival.
- This evidence shows that high levels of serum miR-203 expression may serve as a biomarker for use in predicting both CRC metastasis as well as prognosis.
- miR-203 can be used as a diagnostic and prognostic biomarker.
- high serum miR-203 expression is significantly associated with poor survival and metastasis progression in CRC patients. More importantly, miR-203 appears to be released from CRC cells which have metastasized and found in the circulation. Finally, detection of serum miR-203 may be used to identify patients with metastatic CRC.
- the expression of one or more biomarkers may be measured in a sample of cells or non-cell samples (e.g., biological fluids) from a subject with cancer or suspected of having cancer.
- the type and classification of the cancer can and will vary.
- the cancer may be an early stage cancer, i.e., stage I or stage II, or it may be a late stage cancer, i.e., stage III or stage IV.
- the cancer may be a cancer of the colon or rectum.
- cancers include, but are not limited to, anal cancer, bladder cancer, bone cancer, brain cancer, breast cancer, cervical cancer, duodenal cancer, endometrial cancer, eye cancer, gallbladder cancer, head and neck cancer, liver cancer, larynx cancer, non-small cell lung cancer, small cell lung cancer, lymphomas, melanoma, mouth cancer, ovarian cancer, pancreatic cancer, penal cancer, prostate cancer, rectal cancer, renal cancer, skin cancer, testicular cancer, thyroid cancer, and vaginal cancer.
- anal cancer bladder cancer, bone cancer, brain cancer, breast cancer, cervical cancer, duodenal cancer, endometrial cancer, eye cancer, gallbladder cancer, head and neck cancer, liver cancer, larynx cancer, non-small cell lung cancer, small cell lung cancer, lymphomas, melanoma, mouth cancer, ovarian cancer, pancreatic cancer, penal cancer, prostate cancer, rectal cancer, renal cancer, skin cancer, testicular cancer, thyroid cancer, and vaginal cancer.
- a sample is obtained from the subject under study.
- the sample of cells or tissue sample will be obtained from the subject with cancer by biopsy or surgical resection.
- the type of biopsy can and will vary, depending upon the location and nature of the cancer.
- said sample is a tumor tissue sample or opinion thereof.
- said tumor tissue sample is a colorectal tissue sample from a patient suffering from colorectal cancer.
- Said sample can be obtained by conventional methods, e.g., biopsy, by using methods well known to those of ordinary skill in the related medical arts.
- Methods for obtaining the sample from the biopsy include gross apportioning of a mass, or microdissection or other art-known cell-separation methods.
- Tumor cells can additionally be obtained from fine needle aspiration cytology.
- samples can be obtained from the subject without the assistance of a medical professional; such as the samples can be obtained in the privacy of the subject's home.
- samples that can be procured without the assistance of a medical professional includes, but is not limited to fecal samples, urine samples, blood samples (e.g., whole blood samples), buccal samples, and salvia samples.
- Samples can be obtained from subjects previously diagnosed or not with colorectal cancer, or from subjects who are receiving or have previously received anti-colorectal cancer treatment. In a particular embodiment, samples can be obtained from patients who have not previously received any anti-colorectal cancer treatment.
- these can be formalin-fixed and paraffin-embedded or first frozen and then embedded in a cryosolidifiable medium, such as OCT-Compound, through immersion in a highly cryogenic medium that allows for rapid freeze.
- a cryosolidifiable medium such as OCT-Compound
- the expression levels may be determined using nucleic acids obtained from as fresh tissue from a biopsy or fine needle aspiration cytology.
- tissue samples are envisaged, such a formalin-fixed, paraffin-embedded tissue sample depending on their availability.
- Fixed and paraffin-embedded tissue samples are broadly used storable or archival tissue samples in the field of oncology.
- Nucleic acid may be isolated from an archival pathological sample or biopsy sample which is first deparaffmized.
- An exemplary deparaffmization method involves washing the paraffmized sample with an organic solvent, such as xylene, for example.
- Deparaffmized samples can be rehydrated with an aqueous solution of a lower alcohol. Suitable lower alcohols, for example include, methanol, ethanol, propanols, and butanols.
- Deparaffmized samples may be rehydrated with successive washes with lower alcoholic solutions of decreasing concentration, for example.
- the sample is simultaneously deparaffmized and rehydrated.
- the sample is then lysed and nucleic acid is extracted from the sample.
- tissue selected for fixation and paraffin embedding can be fixed in 10% buffered formalin for 16 hours to 48 hours. After this period of time, said tissue will be embedded in paraffin following conventional techniques. Nevertheless, nucleic acid quality issues are especially delicate when analyzing formalin-fixed tissue samples.
- the expression levels may be determined using nucleic acids obtained from a fecal sample, urine sample, blood sample, serum sample, biopsy tissue sample or fine needle aspiration cytology.
- the sample size required for analysis may range from 1, 10, 50, 100, 200, 300, 500, 1,000, 5,000, 10,000, to 50,000 or more cells.
- the appropriate sample size may be determined based on the cellular composition and condition of the sample (e.g., fecal, blood, urine, serum and/or salvia), biopsy or cytology, and the standard preparative steps for this determination and subsequent isolation of the nucleic acid for use in the invention are well known to one of ordinary skill in the mi.
- a sample of cells, tissue, or fluid may be removed by needle aspiration biopsy.
- a fine needle attached to a syringe may be inserted through the skin and into the organ or tissue of interest.
- the needle may be guided to the region of interest using ultrasound or computed tomography (CT) imaging.
- CT computed tomography
- a vacuum is created with the syringe such that cells or fluid may be sucked through the needle and collected in the syringe.
- a sample of cells or tissue may also be removed by incisional or core biopsy. For this, a cone, a cylinder, or a tiny bit of tissue is removed from the region of interest.
- CT imaging, ultrasound, or an endoscope is generally used to guide this type of biopsy.
- the entire cancerous lesion may be removed by excisional biopsy or surgical resection.
- a sample of cells or sample of tissue may be processed for the isolation of nucleic acids using techniques well known in the art and disclosed in standard molecular biology reference books, such as Ausubel et ah, (2003) Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY.
- a sample of tissue may also be stored in RNAIater (Ambion; Austin TX) or flash frozen and stored at -80 °C for later use.
- the biopsied tissue sample may also be fixed with a fixative, such as formaldehyde, paraformaldehyde, or acetic acid/ethanol.
- the fixed tissue sample may be embedded in wax (paraffin) or a plastic resin.
- the embedded tissue sample (or frozen tissue sample) may be cut into thin sections.
- Nucleic acids may also be extracted from a fixed or wax- embedded tissue sample.
- miRNA of a wide variety of samples can be analyzed using arrays, miRNA probes, or array technology. While endogenous miRNA is contemplated for use with compositions and methods disclosed herein, recombinant miRNA - including nucleic acids that are complementary or identical to endogenous miRNA or precursor miRNA - can also be handled and analyzed as described herein. Samples may be biological samples, in which case, they can be from biopsy, fine needle aspirates, exfoliates, fecal, urine, blood, tissue, organs, semen, saliva, tears, other bodily fluid, hair follicles, skin, or any sample containing or constituting biological cells.
- samples may be, but are not limited to, fresh, frozen, fixed, formalin fixed, paraffin embedded, or formalin fixed and paraffin embedded.
- the sample may not be a biological sample, but a chemical mixture, such as a cell- free reaction mixture (which may contain one or more biological enzymes).
- the subject with cancer will generally be a mammalian subject.
- Mammals may include primates, livestock animals, and companion animals. Primates may include humans, New World monkeys, Old World monkeys, gibbons, and great apes.
- Livestock animals may include horses, cows, goats, sheep, deer (including reindeer) and pigs.
- Companion animals may include dogs, cats, rabbits, and rodents (including mice, rats, and guinea pigs).
- the subject is a human.
- samples may be obtained from a subject based on the results of such a cyto logical analysis.
- a cancer diagnosis may include an examination of a subject by a physician, nurse or other medical professional. The examination may be part of a routine examination, or the examination may be due to a specific complaint.
- a specific complaint may include but is not limited to: pain, illness, anticipation of illness, presence of a suspicious lump or mass, a disease, or a condition.
- the subjects may be suspected of having or be previously determined to have a cancer, a tumor, a pre-cancer, or any other disease.
- the subject may or may not be aware of the disease or condition.
- the subject may be referred to a specialist such as an oncologist, surgeon, or endocrinologist.
- the specialist may likewise obtain a biological sample for testing or refer the individual to a testing center or laboratory for submission of the biological sample.
- the medical professional may refer the subject to a testing center or laboratory for submission of the biological sample.
- the subject may provide the sample.
- a molecular profiling business may obtain the sample.
- methods involve obtaining a sample from a subject.
- the term subject may refer to an animal (for example a mammal), including but not limited to humans, non-human primates, rodents, dogs, or pigs.
- the methods of obtaining provided herein include methods of biopsy such as fine needle aspiration, core needle biopsy, vacuum assisted biopsy, incisional biopsy, excisional biopsy, punch biopsy, shave biopsy or skin biopsy.
- the sample is obtained from a biopsy from colorectal tissue by any of the biopsy methods previously mentioned.
- the sample may be obtained from any of the tissues provided herein that include but are not limited to gall bladder, skin, heart, lung, breast, pancreas, liver, muscle, kidney, smooth muscle, bladder, colon, intestine, brain, prostate, esophagus, or thyroid tissue.
- the sample may be obtained from any other source including but not limited to blood, sweat, hair follicle, buccal tissue, tears, menses, feces, or saliva.
- the sample is obtained from cystic fluid or fluid derived from a tumor or neoplasm.
- the cyst, tumor or neoplasm is colorectal.
- any medical professional such as a doctor, nurse or medical technician may obtain a biological sample for testing.
- the biological sample can be obtained without the assistance of a medical professional.
- a sample may include but is not limited to, tissue, cells, or biological material from cells or derived from cells of a subject.
- the biological sample may be a heterogeneous or homogeneous population of cells or tissues.
- the biological sample may be obtained using any method known to the art that can provide a sample suitable for the analytical methods described herein.
- the sample may be obtained by non-invasive methods including but not limited to: scraping of the skin or cervix, swabbing of the cheek, saliva collection, urine collection, feces collection, collection of menses, tears, or semen.
- the sample may be obtained by methods known in the art.
- the samples are obtained by biopsy.
- the sample is obtained by swabbing, scraping, phlebotomy, or any other methods known in the art.
- the sample may be obtained, stored, or transported using components of a kit of the present methods.
- multiple samples such as multiple colorectal samples may be obtained for diagnosis by the methods described herein.
- multiple samples such as one or more samples from one tissue type (for example colon) and one or more samples from another tissue (for example buccal) may be obtained for diagnosis by the methods.
- multiple samples such as one or more samples from one tissue type (e.g.
- samples from another tissue may be obtained at the same or different times.
- Samples may be obtained at different times are stored and/or analyzed by different methods. For example, a sample may be obtained and analyzed by routine staining methods or any other cytological analysis methods.
- the biological sample may be obtained by a physician, nurse, or other medical professional such as a medical technician, endocrinologist, cytologist, phlebotomist, radiologist, or a pulmonologist.
- the medical professional may indicate the appropriate test or assay to perform on the sample.
- a molecular profiling business may consult on which assays or tests are most appropriately indicated.
- the patient or subject may obtain a biological sample for testing without the assistance of a medical professional, such as obtaining a whole blood sample, a urine sample, a fecal sample, a buccal sample, or a saliva sample.
- the sample is obtained by an invasive procedure including but not limited to: biopsy, needle aspiration, or phlebotomy.
- the method of needle aspiration may further include fine needle aspiration, core needle biopsy, vacuum assisted biopsy, or large core biopsy.
- multiple samples may be obtained by the methods herein to ensure a sufficient amount of biological material.
- the sample is a fine needle aspirate of a colorectal or a suspected colorectal tumor or neoplasm.
- the fine needle aspirate sampling procedure may be guided by the use of an ultrasound, X-ray, or other imaging device.
- the molecular profiling business may obtain the biological sample from a subject directly, from a medical professional, from a third party, or from a kit provided by a molecular profiling business or a third party.
- the biological sample may be obtained by the molecular profiling business after the subject, a medical professional, or a third party acquires and sends the biological sample to the molecular profiling business.
- the molecular profiling business may provide suitable containers, and excipients for storage and transport of the biological sample to the molecular profiling business.
- a medical professional need not be involved in the initial diagnosis or sample acquisition.
- An individual may alternatively obtain a sample through the use of an over the counter (OTC) kit.
- OTC kit may contain a means for obtaining said sample as described herein, a means for storing said sample for inspection, and instructions for proper use of the kit.
- molecular profiling services are included in the price for purchase of the kit. In other cases, the molecular profiling services are billed separately.
- a sample suitable for use by the molecular profiling business may be any material containing tissues, cells, nucleic acids, genes, gene fragments, expression products, gene expression products, or gene expression product fragments of an individual to be tested. Methods for determining sample suitability and/or adequacy are provided.
- a sample may be obtained and prior to analysis by one or more methods described herein, the sample may be stored for a length of time.
- a length of time may include a time interval such as seconds, minutes, hours, days, weeks, months, years or longer.
- the sample obtained from a subject is subdivided prior to the step of storage or further analysis.
- different portions of the sample are subjected to different downstream methods or processes. Such methods or processes may include storage, cytological analysis, integrity tests, nucleic acid extraction, molecular profiling or any combination of these.
- some part of the sample may be stored while another portion of the sample is further processed. Processing may include but is not limited to molecular profiling, cytological staining, gene or gene expression product (RNA or protein) extraction, detection, or quantification, fixation or examination.
- processing may include but is not limited to molecular profiling, cytological staining, gene or gene expression product (RNA or protein) extraction, detection, or quantification, fixation or examination.
- the sample is obtained and stored and subdivided after the step of storage for further analysis such that different portions of the sample are subject to different downstream methods or processes including but not limited to storage, cytological analysis, adequacy tests, nucleic acid extraction, molecular profiling or a combination thereof.
- samples are obtained and analyzed by cytological analysis, and the resulting sample material is further analyzed by one or more molecular profiling methods described herein.
- the samples may be stored between the steps of cytological analysis and the steps of molecular profiling. Samples may be stored upon acquisition to facilitate transport, or to wait for the results of other analyses. In another embodiments, samples may be stored while awaiting instructions a medical professional.
- An acquired sample may be placed in short term or long term storage by placing in a suitable medium, excipient, solution, or container.
- storage may require keeping the sample in a refrigerated, or frozen environment.
- the sample may be quickly frozen prior to storage in a frozen environment.
- the frozen sample may be contacted with a suitable cryopreservation medium or compound.
- cryopreservation mediums or compounds include but are not limited to: glycerol, ethylene glycol, sucrose, or glucose.
- a suitable medium, excipient, or solution may include but is not limited to: hanks salt solution, saline, cellular growth medium, an ammonium salt solution such as ammonium sulphate or ammonium phosphate, or water.
- Suitable concentrations of ammonium salts include solutions of about 0.1 g/ml,
- the medium, excipient, or solution may or may not be sterile.
- the medium, excipient, or solution may contain preservative agents to maintain the sample in an adequate state for subsequent diagnostics or manipulation, or to prevent coagulation.
- Said preservatives may include citrate, ethylene diamine tetraacetic acid, sodium azide, or thimersol.
- the sample may be fixed prior to or during storage by any method known to the art such as using glutaraldehyde, formaldehyde, or methanol.
- the container may be any container suitable for storage and or transport of the biological sample including but not limited to: a cup, a cup with a lid, a tube, a sterile tube, a vacuum tube, a syringe, a bottle, a microscope slide, or any other suitable container.
- the container may or may not be sterile.
- the sample may be stored in a commercial preparation suitable for storage of cells for subsequent cytological analysis such as but not limited to Cytyc ThinPrep, SurePath, or Monoprep.
- the storage temperature may be explicitly defined or defined by a temperature range.
- the sample may be stored at room temperature or at reduced temperatures such as cold temperatures (e.g. between about 20°C. and about 0°C), or freezing temperatures, including for example 0°C, -FC, -2°C, -3°C, -4°C, -5°C, -6°C, -7°C, -8°C, -9°C, -10°C, -12°C, -14°C, -15°C, -16°C, -20°C, -22°C, -25°C, -28°C, -30°C, -35°C, -40°C, -45°C, -50°C, -60°C, -70°C, -80°C,- 100°C, -120°C, -140°C, -180°C, -190°C, -200°C or any ranges or values derivable therein.
- cold temperatures e.g. between about
- the sample may be stored in any condition or environment that allows or achieves the desired temperature condition.
- the samples may be stored in a refrigerator, on ice or a frozen gel pack, in a freezer, in a cryogenic freezer, on dry ice, in liquid nitrogen, or in a vapor phase equilibrated with liquid nitrogen.
- the sample container may be any container suitable for storage and or transport of the biological sample including but not limited to: a cup, a cup with a lid, a tube, a sterile tube, a vacuum tube, a syringe, a bottle, a microscope slide, or any other suitable container.
- the container may or may not be sterile.
- Transport may involve moving or conveyance of a sample to or from a clinic, hospital, doctor's office, or other location to a second location.
- the sample may be stored and/or analyzed by for example, cytological analysis or molecular profiling.
- processing or profiling may begin or take place during transport.
- the sample may be transported to a molecular profiling company in order to perform the analyses described herein.
- the sample may be transported to a laboratory such as a laboratory authorized or otherwise capable of performing the methods described herein, such as a Clinical Laboratory Improvement Amendments (CLIA) laboratory.
- CLIA Clinical Laboratory Improvement Amendments
- the subject may transport the sample.
- Transportation by an individual may include the individual appearing at a molecular profiling business or a designated sample receiving point and providing a sample.
- Providing of the sample may involve any of the techniques of sample acquisition described herein, or the sample may have already have been acquired and stored in a suitable container.
- the sample may be transported to a molecular profiling business using a courier service, the postal service, a shipping service, or any method capable of transporting the sample in a suitable manner.
- the sample may be provided to a molecular profiling business by a third party testing laboratory (e.g. a cytology lab).
- the sample may be provided to a molecular profiling business by the subject's primary care physician, endocrinologist or other medical professional.
- the cost of transport may be billed to the individual, medical provider, or insurance provider.
- the molecular profiling business may begin analysis of the sample immediately upon receipt, or may store the sample in any manner described herein. The method of storage may or may not be the same as chosen prior to receipt of the sample by the molecular profiling business.
- the sample may be transported in any medium or excipient including any medium or excipient provided herein suitable for storing the sample such as a cryopreservation medium or a liquid based cytology preparation.
- the sample may be transported frozen or refrigerated such as at any of the suitable sample storage temperatures provided herein.
- the sample may be assayed using a variety of routine analyses known to the art such as cytological assays, and genomic analysis by a molecular profiling business, a representative or licensee thereof, a medical professional, researcher, or a third party laboratory or testing center (e.g. a cytology laboratory).
- cytological assays and genomic analysis by a molecular profiling business, a representative or licensee thereof, a medical professional, researcher, or a third party laboratory or testing center (e.g. a cytology laboratory).
- Such tests may be indicative of cancer, the type of cancer, any other disease or condition, the presence of disease markers, or the absence of cancer, diseases, conditions, or disease markers.
- the tests may take the form of cytological examination including microscopic examination as described below.
- the tests may involve the use of one or more cytological stains.
- the biological material may be manipulated or prepared for the test prior to administration of the test by any suitable method known to the art for biological sample preparation.
- the specific assay performed may be determined by the molecular profiling company, the physician who ordered the test, or a third party such as a consulting medical professional, cytology laboratory, the subject from whom the sample derives, or an insurance provider.
- the specific assay may be chosen based on the likelihood of obtaining a definite diagnosis, the cost of the assay, the speed of the assay, or the suitability of the assay to the type of material provided.
- sample acquisition concurrent with sample acquisition, sample storage or sample analysis the sample may be subjected to tests or examination that detail or reveal the integrity of the sample for use in the compositions or methods described herein.
- an integrity test a sample may be determined to be adequate or inadequate for further analysis.
- sample integrity tests may pertain to the quality, integrity or adequacy of cells and or tissue in the sample.
- Metrics employed to determine quality, integrity or adequacy may involve but are not limited to cell number tests, cell viability tests, nuclear content tests, genetic content tests, biochemical assays, cell mass tests, cell volume tests, PCR tests, Q-PCR tests, RT-PCR tests, immunochemical analysis, histochemical analysis, microscopic analysis or visual analysis.
- sample integrity may be ascertained by tests that measure nucleic acid content or integrity.
- Nucleic acid content tests may measure DNA content, RNA content or a some mixture of DNA or RNA.
- nucleic acids are extracted or purified from other cellular components prior to a nucleic acid content test.
- nucleic acid specific dyes are used to assay nucleic acid integrity.
- spectrophotometric or electrophoretic methods may be used to assay nucleic acid integrity.
- sample integrity may be ascertained by tests that measure protein content or integrity. Methods that measure protein content or integrity are well known to those skilled in the art. Such methods include but are not limited to ultraviolet absorbance reading (e.g. 280 nm absorbance readings), cell staining, protein staining or immunocytochemical methods. In some instances tests may be performed in parallel in intact samples or the samples may be divided and tests performed serially or in parallel. [00169] Integrity tests may be performed on small subsets or aliquots of a sample or on the entirety of a sample.
- assays include, but are not limited to, array hybridization, solution hybridization, nucleic amplification, polymerase chain reaction, quantitative PCR, RT-PCR, in situ hybridization, Northern hybridization, hybridization protection assay (HP A) (GenProbe), branched DNA (bDNA) assay (Chiron), rolling circle amplification (RCA), single molecule hybridization detection (US Genomics), Invader assay (ThirdWave Technologies), and/or Oligo Ligation Assay (OLA), hybridization, array analysis, digital PCR, ddPCR (digital droplet PCR), nCounter (nanoString), BEAMing (Beads, Emulsions, Amplifications, and Magnetics) (Inostics), ARMS (Amplification Refractory Mutation Systems), RNA-Seq, TAm-Seg (Tagged-Amplicon deep sequencing), and PAP (P
- Nucleic acids may be isolated using techniques well known to those of skill in the art, though in particular embodiments, methods for isolating small nucleic acid molecules, and/or isolating RNA molecules can be employed.
- Chromatography is a process often used to separate or isolate nucleic acids from protein or from other nucleic acids. Such methods can involve electrophoresis with a gel matrix, filter columns, alcohol precipitation, and/or other chromatography.
- methods generally involve lysing the cells with a chaotropic ⁇ e.g., guanidinium isothiocyanate) and/or detergent ⁇ e.g., N-lauroyl sarcosine) prior to implementing processes for isolating particular populations of RNA.
- a chaotropic e.g., guanidinium isothiocyanate
- detergent e.g., N-lauroyl sarcosine
- a gel matrix may be prepared using polyacrylamide, though agarose can also be used.
- the gels may be graded by concentration or they may be uniform. Plates or tubing can be used to hold the gel matrix for electrophoresis.
- one-dimensional electrophoresis may be employed for the separation of nucleic acids. Plates may be used to prepare a slab gel, while the tubing (glass or rubber, typically) can be used to prepare a tube gel.
- the phrase "tube electrophoresis” refers to the use of a tube or tubing, instead of plates, to form the gel. Materials for implementing tube electrophoresis can be readily prepared by a person of skill in the art or purchased.
- Methods may involve the use of organic solvents and/or alcohol to isolate nucleic acids, particularly miRNA used in methods and compositions disclosed herein. Some embodiments are described in U.S. Patent Application Serial No. 10/667,126, which is hereby incorporated by reference.
- this disclosure provides methods for efficiently isolating small cells
- RNA molecules from cells comprising: adding an alcohol solution to a cell lysate and applying the alcohol/lysate mixture to a solid suppoti before eluting the RNA molecules from the solid support.
- the amount of alcohol added to a cell lysate achieves an alcohol concentration of about 55% to 60%. While different alcohols can be employed, ethanol works well.
- a solid support may be any structure, and it includes beads, filters, and columns, which may include a mineral or polymer support with electronegative groups.
- a glass fiber filter or column may work particularly well for such isolation procedures.
- miRNA isolation processes may include: a) lysing cells in the sample with a lysing solution comprising guanidinium, wherein a lysate with a concentration of at least about 1 M guanidinium is produced; b) extracting miRNA molecules from the lysate with an extraction solution comprising phenol; c) adding to the lysate an alcohol solution for forming a lysate/alcohol mixture, wherein the concentration of alcohol in the mixture is between about 35% to about 70%; d) applying the lysate/alcohol mixture to a solid support; e) eluting the miRNA molecules from the solid support with an ionic solution; and, f) capturing the miRNA molecules.
- the sample is dried down and resuspended in a liquid and volume appropriate for subsequent manipulation.
- nucleic acid polymerization and amplification techniques include reverse transcription (RT), polymerase chain reaction (PCR), real-time PCR (quantitative PCR (q-PCR)), nucleic acid sequence-base amplification (NASBA), ligase chain reaction, multiplex ligatable probe amplification, invader technology (Third Wave), rolling circle amplification, in vitro transcription (IVT), strand displacement amplification, transcription-mediated amplification (TMA), R A (Eberwine) amplification, and other methods that are known to persons skilled in the art.
- more than one amplification method may be used, such as reverse transcription followed by real time PCR (Chen et al., 2005 and/or U.S. Patent Application serial number 11/567,082, filed December 5, 2006, which are incorporated herein by reference in its entirety).
- An exemplary PCR reaction includes multiple amplification steps, or cycles that selectively amplify target nucleic acid species.
- An exemplary reaction includes three steps: a denaturing step in which a target nucleic acid is denatured; an annealing step in which a set of PCR primers (forward and reverse primers) anneal to complementary DNA strands; and an elongation step in which a thermostable DNA polymerase elongates the primers. By repeating these steps multiple times, a DNA fragment is amplified to produce an amplicon, corresponding to the target DNA sequence.
- Exemplary PCR reactions may include 20 or more cycles of denaturation, annealing, and elongation.
- the annealing and elongation steps can be performed concurrently, in which case the cycle contains only two steps. Since mature miRNAs are single stranded, a reverse transcription reaction (which produces a complementary cDNA sequence) is performed prior to PCR reactions. Reverse transcription reactions include the use of, e.g., a RNA-based DNA polymerase (reverse transcriptase) and a primer.
- Reverse transcriptase reverse transcriptase
- a set of primers is used for each target sequence.
- the lengths of the primers depends on many factors, including, but not limited to, the desired hybridization temperature between the primers, the target nucleic acid sequence, and the complexity of the different target nucleic acid sequences to be amplified.
- a primer is about 15 to about 35 nucleotides in length. In other embodiments, a primer is equal to or fewer than 15, 20, 25, 30, or 35 nucleotides in length or any range derivable therein. In additional embodiments, a primer is at least 35 nucleotides in length.
- a forward primer can comprise at least one sequence that anneals to a target miRNA and alternatively can comprise an additional 5' noncomplementary region.
- a reverse primer can be designed to anneal to the complement of a reverse transcribed miRNA. The reverse primer may be independent of the miRNA sequence, and multiple miRNAs may be amplified using the same reverse primer. Alternatively, a reverse primer may be specific for a miRNA.
- two or more miRNAs or nucleic acids are amplified in a single reaction volume or multiple reaction volumes.
- one or more miRNA or nucleic acids may be used as a normalization control or a reference nucleic acid for normalization. Normalization may be performed in separate or the same reaction volumes as other amplification reactions.
- One aspect includes multiplex q-PCR, such as qRT-PCR, which enables simultaneous amplification and quantification of at least one miRNA of interest and at least one reference nucleic acid in one reaction volume by using more than one pair of primers and/or more than one probe.
- the primer pairs may comprise at least one amplification primer that uniquely binds each nucleic acid, and the probes are labeled such that they are distinguishable from one another, thus allowing simultaneous quantification of multiple miRNAs.
- Multiplex qRT-PCR has research and diagnostic uses, including but not limited to detection of miRNAs for diagnostic, prognostic, and therapeutic applications.
- a single combined reaction for q-PCR may be used to: (1) decrease risk of experimenter error, (2) reduce assay-to-assay variability, (3) decrease risk of target or product contamination, and (4) increase assay speed.
- the qRT-PCR reaction may further be combined with the reverse transcription reaction by including both a reverse transcriptase and a DNA- based thermostable DNA polymerase.
- a "hot start" approach may be used to maximize assay performance (U.S. Patents 5,411,876 and 5,985,619).
- the components for a reverse transcriptase reaction and a PCR reaction may be sequestered usmg one or more thermoactivation methods or chemical alteration to improve polymerization efficiency (U.S. Patents 5,550,044, 5,413,924, and 6,403,341).
- a panel of amplification targets may be chosen for real-time RT-PCR quantification.
- the panel of targets includes one or more miRNA described herein. The selection of the panel or targets can be based on the results of microarray expression analyses, such as with mzVVanaTM miRNA Bioarray VI (Ambion), Human miRNA Microarrays (V3) (Agilent), miRLinkTM Arrays (Asuragen), or any other suitable microarray.
- a normalization target is 5S rRNA and others can be included.
- Reverse transcription (RT) reaction components may be assembled on ice prior to the addition of RNA template. Total RNA template may be added and mixed. RT reactions may be incubated in an appropriate PCR System at an appropriate temperature (such as 15-30°C, including all values and ranges there between) for an appropriate time, 15 to 30 minutes or longer, then at a temperature of 35 to 42 to 50°C for 10 to 30 to 60 minutes, and then at 80 to 85 to 95°C for 5 minutes, then placed on wet ice.
- Reverse Transcription reaction components may include nuclease-free water, reverse transcription buffer, dNTP mix, RT Primer, RNase Inhibitor, Reverse Transcriptase, and RNA.
- PCR reactions may be incubated in an PCR system at an elevated temperature (e.g., 95°C) for 1 minute or so, then for a number of cycles of denaturing, annealing, and extension (e.g., 40 cycles of 95°C for 5 seconds and 60°C for 30 seconds). Results can be analyzed, for example, with SDS V2.3 (Applied Biosystems).
- Real-time PCR components may include Nuclease-free water, MgCl 2 , PCR Buffer, dNTP mix, one or more primers, DNA Polymerase, cDNA from RT reaction and one or more detectable label.
- Digital polymerase chain reaction (digital PCR, DigitalPCR, dPCR, or dePCR) is a refinement of conventional polymerase chain reaction methods that can be used to directly quantify and clonally amplify nucleic acids including DNA, cDNA or RNA.
- the key difference between dPCR and traditional PCR lies in the method of measuring nucleic acids amounts, with the former being a more precise method than PCR.
- PCR carries out one reaction per single sample.
- dPCR also carries out a single reaction within a sample, however the sample is separated into a large number of partitions and the reaction is carried out in each partition individually. This separation allows a more reliable collection and sensitive measurement of nucleic acid amounts.
- the method has been demonstrated as useful for studying variations in gene sequences—such as copy number variants and point mutations— and it is routinely used for clonal amplification of samples for "next-generation sequencing.”
- a sample is partitioned so that individual nucleic acid molecules within the sample are localized and concentrated within many separate regions.
- the capture or isolation of individual nucleic acid molecules has been effected in micro well plates, capillaries, the dispersed phase of an emulsion, and arrays of miniaturized chambers, as well as on nucleic acid binding surfaces.
- the partitioning of the sample allows one to estimate the number of different molecules by assuming that the molecule population follows the Poisson distribution. As a result, each part will contain "0" or "1" molecules, or a negative or positive reaction, respectively.
- nucleic acids may be quantified by counting the regions that contain PCR end-product, positive reactions.
- dPCR In conventional PCR, the number of PCR amplification cycles is proportional to the starting copy number. dPCR, however, is not dependent on the number of amplification cycles to determine the initial sample amount, eliminating the reliance on uncertain exponential data to quantify target nucleic acids and therefore provides absolute quantification.
- biomarkers may be measured by a variety of techniques that are well known in the art. In addition to the use of arrays and microarrays, it is contemplated that a number of different assays could be employed to analyze miRNAs, their activities, and their effects.
- Such assays include, but are not limited to, digital color-coded barcode technology analysis, microarray expression profiling, quantitative PCR, reverse transcriptase PCR, reverse transcriptase real-time PCR, quantitative real-time PCR, end-point PCR, multiplex end-point PCR, cold PCR, ice-cold PCR, in situ hybridization, Northern hybridization, hybridization protection assay (HP A), branched DNA (bDNA) assay, rolling circle amplification (RCA), single molecule hybridization detection, invader assay, and/or Bridge Litigation Assay.
- a nucleic acid microarray may be used to quantify the differential expression of a plurality of biomarkers.
- Microarray analysis may be performed using commercially available equipment, following manufacturer's protocols, such as by using the Affymetrix GeneChip® technology (Santa Clara, CA) or the Microarray System from Incyte (Fremont, CA).
- single-stranded nucleic acids ⁇ e.g., miRNAs, cDNAs or oligonucleotides
- the arrayed sequences may be then hybridized with specific nucleic acid probes from the cells of interest.
- Fluorescently labeled cDNA probes may be generated through incorporation of fluorescently labeled deoxynucleotides by reverse transcription of RNA extracted from the cells of interest.
- the RNA may be amplified by in vitro transcription and labeled with a marker, such as biotin.
- the labeled probes may then be hybridized to the immobilized nucleic acids on the microchip under highly stringent conditions. After stringent washing to remove the non-specifically bound probes, the chip may be scanned by confocal laser microscopy or by another detection method, such as a CCD camera.
- the raw fluorescence intensity data in the hybridization files may be preprocessed with the robust multichip average (RMA) algorithm to generate expression values.
- RMA robust multichip average
- Quantitative real-time PCR may also be used to measure the differential expression of a plurality of biomarkers.
- the RNA template is generally reverse transcribed into cDNA, which is then amplified via a PCR reaction.
- the amount of PCR product is followed cycle-by-cycle in real time, which allows for determination of the initial concentrations of miRNA.
- the reaction may be performed in the presence of a fluorescent dye, such as SYBR Green, which binds to double- stranded DNA.
- the reaction may also be performed with a fluorescent reporter probe that is specific for the DNA being amplified.
- a non-limiting example of a fluorescent reporter probe is a TaqMan® probe
- Multiplex qRT-PCR may be performed by using multiple gene-specific reporter probes, each of which contains a different fluorophore. Fluorescence values are recorded during each cycle and represent the amount of product amplified to that point in the amplification reaction.
- qRT-PCR may be performed using a reference standard.
- the ideal reference standard is expressed at a constant level among different tissues, and is unaffected by the experimental treatment.
- the level of miRNA in the original sample or the fold change in expression of each biomarker may be determined using calculations well known in the art.
- Luminex® multiplexing microspheres may also be used to measure the differential expression of a plurality of biomarkers.
- These microscopic polystyrene beads are internally color-coded with fluorescent dyes, such that each bead has a unique spectral signature (of which there are up to 100). Beads with the same signature are tagged with a specific oligonucleotide or specific antibody that will bind the target of interest (i.e., biomarker miRNA).
- the target is also tagged with a fluorescent reporter.
- there are two sources of color one from the bead and the other from the reporter molecule on the target.
- the beads are then incubated with the sample containing the targets, of which up 100 may be detected in one well.
- the small size/surface area of the beads and the three dimensional exposure of the beads to the targets allows for nearly solution-phase kinetics during the binding reaction.
- the captured targets are detected by high-tech fluidics based upon flow cytometry in which lasers excite the internal dyes that identify each bead and also any reporter dye captured during the assay.
- the data from the acquisition files may be converted into expression values using means known in the art.
- In situ hybridization may also be used to measure the differential expression of a plurality of biomarkers.
- This method permits the localization of miRNAs of interest in the cells of a tissue section.
- the tissue may be frozen, or fixed and embedded, and then cut into thin sections, which are arrayed and affixed on a solid surface.
- the tissue sections are incubated with a labeled antisense probe that will hybridize with an miRNA of interest.
- the hybridization and washing steps are generally performed under highly stringent conditions.
- the probe may be labeled with a fluorophore or a small tag (such as biotin or digoxigenin) that may be detected by another protein or antibody, such that the labeled hybrid may be detected and visualized under a microscope.
- each antisense probe has a distinguishable label.
- the hybridized tissue array is generally scanned under a microscope. Because a sample of tissue from a subject with cancer may be heterogeneous, i.e., some cells may be normal and other cells may be cancerous, the percentage of positively stained cells in the tissue may be determined. This measurement, along with a quantification of the intensity of staining, may be used to generate an expression value for each biomarker.
- the number of biomarkers whose expression is measured in a sample of cells from a subject with cancer may vary. Since the risk score is based upon the differential expression of the biomarkers, a higher degree of accuracy should be attained when the expression of more biomarkers is measured; however, a large number of biomarkers in the gene signature would hamper the clinical usefulness.
- the differential expression of at least, at most, or about 20, 15, 10, 8, 6, 5, 4, 3 or 2 biomarkers (or any ranges or values derivable therein) may be measured.
- miR A arrays or miRNA probe arrays which are ordered macroarrays or microarrays of nucleic acid molecules (probes) that are fully or nearly complementary or identical to a plurality of miRNA molecules or precursor miRNA molecules and are positioned on a support or support material in a spatially separated organization.
- Macroarrays are typically sheets of nitrocellulose or nylon upon which probes have been spotted.
- Microarrays position the nucleic acid probes more densely such that up to 10,000 nucleic acid molecules can be fit into a region typically 1 to 4 square centimeters.
- miR A anays or miRNA probe arrays which are ordered macroarrays or microarrays of nucleic acid molecules (probes) that are fully or nearly complementary or identical to a plurality of miRNA molecules or precursor miRNA molecules and that are positioned on a support or support material in a spatially separated organization.
- Macroarrays are typically sheets of nitrocellulose or nylon upon which probes have been spotted.
- Microarrays position the nucleic acid probes more densely such that up to 10,000 nucleic acid molecules can be fit into a region typically 1 to 4 square centimeters.
- Microarrays can be fabricated by spotting nucleic acid molecules, e.g., genes, oligonucleotides, etc., onto substrates or fabricating oligonucleotide sequences in situ on a substrate. Spotted or fabricated nucleic acid molecules can be applied in a high density matrix pattern of up to about 30 non-identical nucleic acid molecules per square centimeter or higher, e.g. up to about 100 or even 1000 per square centimeter. Microarrays typically use coated glass as the solid support, in contrast to the nitrocellulose-based material of filter arrays. By having an ordered array of miRNA-complementing nucleic acid samples, the position of each sample can be tracked and linked to the original sample.
- nucleic acid molecules e.g., genes, oligonucleotides, etc.
- array devices in which a plurality of distinct nucleic acid probes are stably associated with the surface of a solid support are known to those of skill in the art.
- Useful substrates for arrays include nylon, glass, metal, plastic, and silicon.
- Such arrays may vary in a number of different ways, including average probe length, sequence or types of probes, nature of bond between the probe and the array surface, e.g. covalent or non-covalent, and the like.
- the labeling and screening methods are not limited by with respect to any parameter except that the probes detect miR A; consequently, methods and compositions may be used with a variety of different types of miRNA arrays.
- the arrays can be high density arrays, such that they contain 2, 20, 25, 50, 80, 100, or more, or any integer derivable therein, different probes. It is contemplated that they may contain 1000, 16,000, 65,000, 250,000 or 1,000,000 or more, or any interger or range derivable therein, different probes.
- the probes can be directed to targets in one or more different organisms or cell types.
- the oligonucleotide probes may range from 5 to 50, 5 to 45, 10 to 40, 9 to 34, or 15 to 40 nucleotides in length. In certain embodiments, the oligonucleotide probes are 5, 10, 15, 20, 25, 30, 35, 40 nucleotides in length, including all integers and ranges there between.
- the large number of different probes can occupy a relatively small area providing a high density array having a probe density of generally greater than about 60, 100, 600, 1000, 5,000, 10,000, 40,000, 100,000, or 400,000 different oligonucleotide probes per cm 2 .
- the surface area of the array can be about or less than about 1, 1.6, 2, 3, 4, 5, 6, 7, 8, 9, or 10 cm 2 .
- the population of target nucleic acids may be contacted with the array or probes under hybridization conditions, where such conditions can be adjusted, as desired, to provide for an optimum level of specificity in view of the particular assay being performed.
- Suitable hybridization conditions are well known to those of skill in the art and reviewed in Sambrook et al. (2001) and WO 95/21944. Of particular interest in many embodiments is the use of stringent conditions during hybridization. Stringent conditions are known to those of skill in the art.
- methods concern miRNA that are directly or indirectly labeled. It is contemplated that miRNA may first be isolated and/or purified prior to labeling. This may achieve a reaction that more efficiently labels the miRNA, as opposed to other RNA in a sample in which the miRNA is not isolated or purified prior to labeling.
- the label is non-radioactive.
- nucleic acids may be labeled by adding labeled nucleotides (one-step process) or adding nucleotides and labeling the added nucleotides (two-step process).
- nucleic acids are labeled by catalytically adding to the nucleic acid an already labeled nucleotide or nucleotides.
- One or more labeled nucleotides can be added to miRNA molecules. See U.S Patent 6,723,509, which is hereby incorporated by reference.
- an unlabeled nucleotide or nucleotides may be catalytically added to a miRNA, and the unlabeled nucleotide is modified with a chemical moiety that enables it to be subsequently labeled.
- the chemical moiety is a reactive amine such that the nucleotide is an amine-modified nucleotide. Examples of amine-modified nucleotides are well known to those of skill in the art, many being commercially available such as from Ambion, Sigma, Jena Bioscience, and TriLink.
- embodiments concern the use of an enzyme capable of using a di- or tri-phosphate ribonucleotide or deoxytibonucleotide as a substrate for its addition to a miRNA. Moreover, in specific embodiments, it involves using a modified di- or tri-phosphate ribonucleotide, which is added to the 3' end of a miRNA.
- the source of the enzyme is not limiting. Examples of sources for the enzymes include yeast, gram- negative bacteria such as E. coli, Lactococcus lactis, and sheep pox virus.
- Enzymes capable of adding such nucleotides include, but are not limited to, poly(A) polymerase, terminal transferase, and polynucleotide phosphorylase.
- a ligase is contemplated as not being the enzyme used to add the label, and instead, a non-ligase enzyme is employed.
- Terminal transferase catalyzes the addition of nucleotides to the 3' terminus of a nucleic acid.
- Polynucleotide phosphorylase can polymerize nucleotide diphosphates without the need for a primer.
- Labels on miRNA or miRNA probes may be colorimetric (includes visible and
- UV spectrum including fluorescent
- luminescent including fluorescent
- enzymatic including radioactive
- positron emitting including radioactive
- the label may be detected directly or indirectly.
- Radioactive labels include 125 I,
- enzymatic labels include alkaline phosphatase, luciferase, horseradish peroxidase, and ⁇ -galactosidase.
- Labels can also be proteins with luminescent properties, e.g., green fluorescent protein and phycoerythrin.
- the colorimetric and fluorescent labels contemplated for use as conjugates include, but are not limited to, Alexa Fluor dyes, BODIPY dyes, such as BODIPY FL; Cascade Blue; Cascade Yellow; coumarin and its derivatives, such as 7-amino-4-methylcoumarin, aminocoumarin and hydroxycoumarin; cyanine dyes, such as Cy3 and Cy5; eosins and erythrosins; fluorescein and its derivatives, such as fluorescein isothiocyanate; macrocyclic chelates of lanthanide ions, such as Quantum DyeTM; Marina Blue; Oregon Green; rhodamine dyes, such as rhodamine red, tetramethylrhodamine and rhodamine 6G; Texas Red; fluorescent energy transfer dyes, such as thiazole orange-ethidium heterodimer; and, TOTAB.
- Alexa Fluor dyes such as BODIPY FL
- Cascade Blue
- dyes include, but are not limited to, those identified above and the following: Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 500, Alexa Fluor 514, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 610, Alexa Fluor 633, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700, and, Alexa Fluor 750; amine-reactive BODIPY dyes, such as BODIPY 493/503, BODIPY 530/550, BODIPY 558/568, BODIPY 564/570, BODIPY 576/589, BODIPY 581/591, BODIPY 630/650, BODIPY 650/655, BODIPY FL, BODIPY R6G, BODIPY TMR, and
- Molecular Probes include, Alexa Fluor 488-5-UTP, Fluorescein- 12-UTP, BODIPY FL-14-UTP, BODIPY TMR-14-UTP, Tetramethylrhodamine-6-UTP, Alexa Fluor 546-14-UTP, Texas Red-5-UTP, and BODIPY TR-14-UTP.
- Other fluorescent ribonucleotides are available from Amersham Biosciences, such as Cy3-UTP and Cy5-UTP.
- Examples of fluorescently labeled deoxyribonucleotides include Dinitrophenyl
- (DNP)-l l-dUTP Cascade Blue-7-dUTP, Alexa Fluor 488-5-dUTP, Fluorescein- 12-dUTP, Oregon Green 488-5-dUTP, BODIPY FL-14-dUTP, Rhodamine Green-5-dUTP, Alexa Fluor 532-5-dUTP, BODIPY TMR-14-dUTP, Tetramethylrhodamine-6-dUTP, Alexa Fluor 546-14- dUTP, Alexa Fluor 568-5-dUTP, Texas Red-12-dUTP, Texas Red-5-dUTP, BODIPY TR-14- dUTP, Alexa Fluor 594-5-dUTP, BODIPY 630/650-14-dUTP, BODIPY 650/665-14-dUTP; Alexa Fluor 488-7-OBEA-dCTP, Alexa Fluor 546-16-OBEA-dCTP, Alexa Fluor 594-7-OBEA- dC
- nucleic acids may be labeled with two different labels.
- FRET fluorescence resonance energy transfer
- the label may not be detectable per se, but indirectly detectable or allowing for the isolation or separation of the targeted nucleic acid.
- the label could be biotin, digoxigenin, polyvalent cations, chelator groups and the other ligands, include ligands for an antibody.
- a number of techniques for visualizing or detecting labeled nucleic acids are readily available. Such techniques include, microscopy, arrays, Fluorometry, Light cyclers or other real time PCR machines, FACS analysis, scintillation counters, Phosphoimagers, Geiger counters, MRI, CAT, antibody-based detection methods (Westerns, immunofluorescence, immunohistochemistry), histochemical techniques, HPLC (Griffey et ah, 1997), spectroscopy, capillary gel electrophoresis (Cummins et ah, 1996), spectroscopy; mass spectroscopy; radiological techniques; and mass balance techniques.
- FRET fluorescent resonance energy transfer
- Methods can be used to detect differences in miRNA expression or levels between two samples, or a sample and a reference ⁇ e.g., a tissue or other biological reference or a digital reference representative of a non-cancerous state).
- a reference e.g., a tissue or other biological reference or a digital reference representative of a non-cancerous state.
- Specifically contemplated applications include identifying and/or quantifying differences between miRNA from a sample that is normal and from a sample that is not normal, between a cancerous condition and a non-cancerous condition, or between two differently treated samples ⁇ e.g., a pretreatment versus a post- treatment sample).
- miRNA may be compared between a sample believed to be susceptible to a particular therapy, disease, or condition and one believed to be not susceptible or resistant to that therapy, disease, or condition.
- a sample that is not normal is one exhibiting phenotypic trait(s) of a disease or condition or one believed to be not normal with respect to that disease or condition. It may be compared to a cell that is normal relative to that disease or condition.
- Phenotypic traits include symptoms of a disease or condition of which a component is or may or may not be genetic or caused by a hyperproliferative or neoplastic cell or cells, such as nodules or tumors.
- Phenotypic traits also include characteristics such as longevity, morbidity, appearance (e.g., baldness, obesity), strength, speed, endurance, fertility, susceptibility or receptivity to particular drugs or therapeutic treatments (drug efficacy), and risk of drug toxicity.
- miR A profiles may be generated to evaluate and correlate those profiles with pharmacokinetics.
- miRNA profiles may be created and evaluated for patient tumor and blood samples prior to the patient's being treated or during treatment to determine if there are miRNAs whose expression correlates with the outcome of treatment. Identification of differential miRNAs can lead to a diagnostic assay involving them that can be used to evaluate tumor and/or blood samples to determine what drug regimen the patient should be provided. In addition, it can be used to identify or select patients suitable for a particular clinical trial. If a miRNA profile is determined to be correlated with drug efficacy or drug toxicity that determination may be relevant to whether that patient is an appropriate patient for receiving the drug or for a particular dosage of the drug.
- blood samples from patients can be evaluated to identify a disease or a condition based on miRNA levels, such as primary CRC, dysplasia or a metastatic disease.
- a diagnostic assay can be created based on the profiles that doctors can use to identify individuals with a disease or a prognostic assay to determine or identify those individuals that who are at risk to develop a disease or condition such as metastasis.
- treatments can be designed based on miRNA profiling. Examples of such methods and compositions are described in the U.S. Provisional Patent Application entitled "Methods and Compositions Involving miRNA and miRNA Inhibitor Molecules" filed on May 23, 2005, which is hereby incorporated by reference in its entirety.
- this invention entails measuring expression of one or more biomarkers in a sample of cells from a subject with cancer.
- the expression information may be obtained by testing cancer samples by a lab, a technician, a device, or a clinician.
- the pattern or signature of expression in each sample may then be used to generate a risk score for cancer prognosis or classification, such as predicting cancer survival or recurrence.
- the level of expression of a biomarker may be increased or decreased in a subject relative to other subjects with cancer.
- the expression of a biomarker may be higher in long-term survivors than in short-term survivors.
- the expression of a biomarker may be higher in short-term survivors than in long-term survivors.
- embodiments can be used to evaluate differences between stages of disease, such as between hyperplasia, dysplasia, neoplasia, precancer and cancer, or between a primary tumor and a metastasized tumor.
- Neoplastic cells and “neoplasia” may be used interchangeably and refer to cells which exhibit relatively autonomous growth, so that they exhibit an aberrant growth phenotype characterized by a significant loss of control of cell proliferation.
- Neoplastic cells can be malignant or benign.
- a neoplasia includes both dysplasia and cancer.
- Neoplasms may be benign, pre-malignant (carcinoma in situ or dysplasia) or malignant (cancer).
- Neoplastic cells may form a lump (i.e., a tumor) or not.
- Dysplasia may be used when the cellular abnormality is restricted to the originating tissue, as in the case of an early, in-situ neoplasm. Dysplasia may be indicative of an early neoplastic process.
- cancer may refer to a malignant neoplasm, including a broad group of various diseases involving unregulated cell growth.
- Metastasis may refer to the spread of a cancer from one organ or part to another non-adjacent organ or part.
- the new occurrences of disease thus generated may be referred to as metastases.
- Cancers that may be evaluated by the disclosed methods and compositions include cancer cells particularly from the pancreas, including pancreatic ductal adenocarcinoma (PDAC), but may also include cells and cancer cells from the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestine, gum, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, testis, tongue, or uterus.
- PDAC pancreatic ductal adenocarcinoma
- the cancer may specifically be of the following histological type, though it is not limited to these: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcmoma; combined hepatocellular carcmoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcmoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcmoma; carcinoid tumor, malignant; branchiolo-alveolar adenocarcinoma; pa
- the disclosed methods and compositions can be used to evaluate differences between stages of disease, such as between hyperplasia, neoplasia, pre-cancer and cancer, or between a primary tumor and a metastasized tumor.
- samples that have differences in the activity of certain pathways may also be compared. These pathways include the following and those involving the following factors: antibody response, apoptosis, calcium/NFAT signaling, cell cycle, cell migration, cell adhesion, cell division, cytokines and cytokine receptors, drug metabolism, growth factors and growth factor receptors, inflammatory response, insulin signaling, NF K -B signaling, angiogenesis, adipogenesis, cell adhesion, viral infection, bacterial infection, senescence, motility, glucose transport, stress response, oxidation, aging, telomere extension, telomere shortening, neural transmission, blood clotting, stem cell differentiation, G- Protein Coupled Receptor (GPCR) signaling, and p53 activation.
- GPCR G- Protein Coupled Receptor
- Cellular pathways that may be profiled also include but are not limited to the following: any adhesion or motility pathway including but not limited to those involving cyclic AMP, protein kinase A, G-protein couple receptors, adenylyl cyclase, L-selectin, E-selectin, PEC AM, VCAM-1, a-actinin, paxillin, cadherins, AKT, integrin- a, integrin- ⁇ , RAF-1, ER , PI- 3 kinase, vinculin, matrix metalloproteinases, Rho GTPases, p85, trefoil factors, profilin, FAK, MAP kinase, Ras, caveolin, calpain-1, calpain-2, epidermal growth factor receptor, ICAM-1, ICAM-2, cofilin, actin, gelsolin, RhoA, RACl, myosin light chain kinase, platelet
- a variety of different models can be employed to evaluate expression levels and/or other comparative values based on expression levels of miRNAs (or their precursors or targets).
- One model is a logistic regression model (see the Wikipedia entry on the World Wide Web at en.wikipedia.com, which is hereby incorporated by reference).
- a scoring algorithm comprises a method selected from the group consisting of: Linear Discriminate Analysis (LDA), Significance Analysis of Microarrays, Tree Harvesting, CART, MARS, Self Organizing Maps, Frequent Item Set, Bayesian networks, Prediction Analysis of Microarray (PAM), SMO, Simple Logistic Regression, Logistic Regression, Multilayer Perceptron, Bayes Net, Naive Bayes, Naive Bayes Simple, Naive Bayes Up, IB1, Ibk, Kstar, LWL, AdaBoost, ClassViaRegression, Decorate, Multiclass Classifier, Random Committee, j48, LMT, NBTree, Part, Random Forest, Ordinal Classifier, Sparse Linear Programming (SPLP), Sparse Logistic Regression (SPLR), Elastic NET, Support Vector Machine, Prediction of Residual Enor Sum of Squares
- LDA Linear Discriminate Analysis
- SPLP Sparse Linear Programming
- SPLR Sparse Logistic Regression
- LDA linear discriminate analysis
- Models may take into account one or more different comparison values or they may also take into account differential expression of one or more additional biomarkers.
- a diagnostic or risk score may be based on 1, 2, 3 ,4 ,5 ,6, 7, 8 or more biomarkers (or any range derivable therein), but in some embodiments, it takes into account additionally or alternatively, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more miRNA expression levels (or any range derivable therein), wherein the miRNA expression level delectably differs between metastatic cells and cells that are not metastatic.
- a score is prepared.
- the score may involve numbers such as 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, (or any range or a subset therein) in some embodiments.
- RS Risk Score
- the biomarkers are related to cancer diagnosis or prognosis, for example, prediction of dysplasia, early stage CRC, primary CRC, metastasis risk, survival, recurrence, or therapy response.
- the differential patterns of expression of a plurality of these biomarkers may be used to predict the survival outcome of a subject with cancer. Certain biomarkers tend to be over-expressed in long-term survivors, whereas other biomarkers tend to be overexpressed in short-term survivors.
- the unique pattern of expression of a plurality of biomarkers in a subject i.e., the gene signature
- Subjects with a high risk score may have a short survival time (e.g., less than about 2 years) after surgical resection.
- Subjects with a low risk score may have a longer survival time (e.g., more than about 3 years) after resection.
- biomarkers associated with early stage detection of CRC may be used to identify subjects at risk associated with colorectal cancer or and/or identify subjects with a favorable or poor prognosis.
- certain aspects also included metastasis-associated biomarkers that may be used for identifying cells with metastatic potential within the primary tumor and hope for improving the prognosis of these metastatic-associated cancers. Additionally, by identifying the metastasis-associated biomarkers whose expression is changed in metastasis offers potential targets to inhibit metastasis.
- the expression of each biomarker may be converted into an expression value. These expression values then will be used to calculate a risk score of survival for a subject with cancer using statistical methods well known in the art.
- the risk scores may be calculated using a principal components analysis.
- the risk scores may also be calculated using a partial Cox regression analysis.
- the risk scores may be calculated using a univariate Cox regression analysis.
- the scores generated may be used to classify patients into high or low risk signature, wherein a high risk score is associated with a poor diagnosis, metastasis risk or prognosis, such as a short survival time or a poorer survival, and a low risk score is associated with a good diagnosis, metastasis risk, or prognosis, such as a long survival time or a better survival.
- the cut-off value may be derived from a control group of cancer patients as a median risk score.
- a tissue sample may be collected from a subject with a cancer, for example, a colorectal cancer.
- the collection step may comprise surgical resection.
- the sample of tissue may be stored in RNA later or flash frozen, such that RNA may be isolated at a later date.
- miRNA may be isolated from the tissue and used to generate labeled probes for a nucleic acid microarray analysis.
- the miRNA may also be used as template for qRT-PCR in which the expression of a plurality of biomarkers is analyzed.
- the expression data generated may be used to derive a risk score, e.g., using the Cox regression classification method.
- the risk score may be used to predict whether the subject will be have a risk for developing cancer or metastasis or a short-term or a long-term cancer survivor.
- Methods may involve the determination or selection of an appropriate cancer
- management regimen and predicting the outcome of the same.
- management regimen refers to a management plan that specifies the type of examination, screening, diagnosis, surveillance, care, and treatment (such as dosage, schedule and/or duration of a treatment) provided to a subject in need thereof (e.g., a subject diagnosed with cancer).
- the selected treatment regimen can be an aggressive one which is expected to result in the best clinical outcome (e.g., complete cure of the disease) or a more moderate one which may relieve symptoms of the disease yet results in incomplete cure of the disease.
- the type of treatment can include a surgical intervention, administration of a therapeutic drug, an exposure to radiation therapy and/or any combination thereof.
- the dosage, schedule and duration of treatment can vary, depending on the severity of disease and the selected type of treatment, and those of skill in the art are capable of adjusting the type of treatment with the dosage, schedule and duration of treatment.
- Biomarkers and a new "Risk Score" system that can predict the likelihood of tumor or cancer recurrence or overall survival in cancer patients can be used to identify patients who will get benefit of conventional single or combined modality therapy before treatment begins.
- the invention can identify those patients who do not get much benefit from such conventional single or combined modality therapy and can offer them alternative treatment(s).
- further cancer or metastasis examination or screening such as fecal occult blood testing, flexible sigmoidoscopy and colonoscopy for colorectal cancer, or further diagnosis such as contrast enhanced computed tomography (CT), positron emission tomography-CT (PET-CT), and magnetic resonance imaging (MRI) may be performed for the detection of CRC or cancer metastasis in patients determined to at high risk based on the miRNA expression levels.
- CT contrast enhanced computed tomography
- PET-CT positron emission tomography-CT
- MRI magnetic resonance imaging
- Non-limiting examples of screening tests include fecal occult blood testing, flexible sigmoidoscopy and colonoscopy.
- Sigmoidoscopy may not screen the right side of the colon where 42% of malignancies are found.
- Virtual colonoscopy via a CT scan appears as good as standard colonoscopy for detecting cancers and large adenomas but is expensive, associated with radiation exposure, and cannot remove any detected abnormal growths like standard colonoscopy can.
- a new screening method is the M2-PK Test.
- the enzyme biomarker M2-PK has been identified as a key enzyme in colorectal cancers and polyps. M2-PK does not depend on blood in the stool and is specifically related to changes in the tumour metabolism. It does not require any special preparation prior to testing.
- M2-PK features a high sensitivity for colorectal cancer and polyps and is able to detect bleeding and non- bleeding colorectal cancer and polyps. In the event of a positive result people would be asked to undergo further examination e.g. colonoscopy.
- Fecal occult blood testing of the stool may be recommended every two years and can be either guaiac based or immunochemical. For those at high risk, screenings may be more frequent or more early as compared with recommended guidelines for people with average risk. For people with average risk who have had a high-quality colonoscopy with normal results, the American Gastroenterological Association does not recommend any type of screening in the 10 years following the colonoscopy. For people over 75 or those with a life expectancy of less than I 0 years, screening may not be recommended.
- conventional cancer therapy or therapy for early cancer may be applied to a subject wherein the subject is identified or reported as having a good prognosis or low risk of metastasis based on the assessment of the biomarkers as disclosed.
- normal, low or moderate surveillance may be provided for patients with a favorable prognosis or biomarker profile or low risk of cancer or metastasis.
- At least an alternative cancer therapy or metastasis therapy or care may be prescribed, as used alone or in combination with conventional cancer therapy, if a poor prognosis or high risk of metastasis is determined by the disclosed methods or kits.
- intensive or aggressive surveillance may be provided for patients with an unfavorable prognosis or biomarker profile or high risk of cancer or metastasis.
- Conventional cancer therapies include one or more selected from the group of chemical or radiation based treatments and surgery.
- Chemotherapies include, for example, cisplatin (CDDP), carboplatin, procarbazine, mechlorethamine, cyclophosphamide, camptothecin, ifosfamide, melphalan, chlorambucil, busulfan, nitrosurea, dactinomycin, daunorubicin, doxorubicin, bleomycin, plicomycin, mitomycin, etoposide (VP 16), tamoxifen, raloxifene, estrogen receptor binding agents, taxol, gemcitabien, navelbine, farnesyl-protein tansferase inhibitors, transplatinum, 5-fluorouracil, vincristin, vinblastin and methotrexate, or any analog or derivative variant of the foregoing.
- CDDP cisplatin
- carboplatin carboplatin
- Radioisotopes Radiation therapy that cause DNA damage and have been used extensively include what are commonly known as ⁇ -rays, X-rays, and/or the directed delivery of radioisotopes to tumor cells. Other forms of DNA damaging factors are also contemplated such as microwaves and UV-irradiation. It is most likely that all of these factors effect a broad range of damage on DNA, on the precursors of DNA, on the replication and repair of DNA, and on the assembly and maintenance of chromosomes. Dosage ranges for X-rays range from daily doses of 50 to 200 roentgens for prolonged periods of time (3 to 4 wk), to single doses of 2000 to 6000 roentgens. Dosage ranges for radioisotopes vary widely, and depend on the half-life of the isotope, the strength and type of radiation emitted, and the uptake by the neoplastic cells.
- contacted and “exposed,” when applied to a cell are used herein to describe the process by which a therapeutic construct and a chemotherapeutic or radiotherapeutic agent are delivered to a target cell or are placed in direct juxtaposition with the target cell.
- both agents are delivered to a cell in a combined amount effective to kill the cell or prevent it from dividing.
- Curative surgery is a cancer treatment that may be used in conjunction with other therapies, such as the treatment of the present invention, chemotherapy, radiotherapy, hormonal therapy, gene therapy, immunotherapy and/or alternative therapies.
- Curative surgery includes resection m which all or part of cancerous tissue is physically removed, excised, and/or destroyed.
- Tumor resection refers to physical removal of at least part of a tumor.
- treatment by surgery includes laser surgery, cryosurgery, electrosurgery, and microscopically controlled surgery (Mohs' surgery). It is further contemplated that the present invention may be used in conjunction with removal of superficial cancers, precancers, or incidental amounts of normal tissue.
- a cavity may be formed in the body.
- Treatment may be accomplished by perfusion, direct injection or local application of the area with an additional anti-cancer therapy.
- Such treatment may be repeated, for example, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5 weeks or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months.
- These treatments may be of varying dosages as well.
- Alternative cancer therapy include any cancer therapy other than surgery, chemotherapy and radiation therapy in the present invention, such as immunotherapy, gene therapy, hormonal therapy or a combination thereof.
- Subjects identified with poor prognosis using the present methods may not have favorable response to conventional treatment(s) alone and may be prescribed or administered one or more alternative cancer therapy per se or in combination with one or more conventional treatments.
- Immunotherapeutics generally, rely on the use of immune effector cells and molecules to target and destroy cancer cells.
- the immune effector may be, for example, an antibody specific for some marker on the surface of a tumor cell.
- the antibody alone may serve as an effector of therapy or it may recruit other cells to actually effect cell killing.
- the antibody also may be conjugated to a drug or toxin (chemotherapeutic, radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.) and serve merely as a targeting agent.
- the effector may be a lymphocyte carrying a surface molecule that interacts, either directly or indirectly, with a tumor cell target.
- Various effector cells include cytotoxic T cells and NK cells.
- Gene therapy is the insertion of polynucleotides, including DNA or RNA, into an individual's cells and tissues to treat a disease.
- Antisense therapy is also a form of gene therapy in the present invention.
- a therapeutic polynucleotide may be administered before, after, or at the same time of a first cancer therapy. Delivery of a vector encoding a variety of proteins is encompassed within the invention. For example, cellular expression of the exogenous tumor suppressor oncogenes would exert their function to inhibit excessive cellular proliferation, such as p53, pl6 and C -CAM.
- Additional agents to be used to improve the therapeutic efficacy of treatment include immunomodulatory agents, agents that affect the upregulation of cell surface receptors and GAP junctions, cytostatic and differentiation agents, inhibitors of cell adhesion, or agents that increase the sensitivity of the hyperproliferative cells to apoptotic inducers.
- Immunomodulatory agents include tumor necrosis factor; interferon alpha, beta, and gamma; IL- 2 and other cytokines; F42K and other cytokine analogs; or MIP-1, MIP-1 beta, MCP-1, RANTES, and other chemokines.
- cell surface receptors or their ligands such as Fas / Fas ligand, DR4 or DR5 / TRAIL would potentiate the apoptotic inducing abilities of the present invention by establishment of an autocrine or paracrine effect on hyperproliferative cells. Increases intercellular signaling by elevating the number of GAP junctions would increase the anti-hyperproliferative effects on the neighboring hyperproliferative cell population.
- cytostatic or differentiation agents can be used in combination with the present invention to improve the anti-hyperproliferative efficacy of the treatments.
- Inhibitors of cell adhesion are contemplated to improve the efficacy of the present invention.
- cell adhesion inhibitors are focal adhesion kinase (FAKs) inhibitors and Lovastatin. It is further contemplated that other agents that increase the sensitivity of a hyperproliferative cell to apoptosis, such as the antibody c225, could be used in combination with the present invention to improve the treatment efficacy.
- FAKs focal adhesion kinase
- Lovastatin Lovastatin
- Hormonal therapy may also be used in the present invention or in combination with any other cancer therapy previously described.
- the use of hormones may be employed in the treatment of certain cancers such as breast, prostate, ovarian, or cervical cancer to lower the level or block the effects of certain hormones such as testosterone or estrogen. This treatment is often used in combination with at least one other cancer therapy as a treatment option or to reduce the risk of metastases.
- a cancer Once a cancer has metastasized or is determined to be at high risk for metastasis, it may still be treated with radiosurgery, chemotherapy, radiation therapy, biological therapy, hormone therapy, surgery, or a combination of these interventions ("multimodal therapy").
- multimodal therapy The choice of treatment depends on a large number of factors, including the type of primary cancer, the size and location of the metastases, the patient's age and general health, and the types of treatments used previously, among others.
- the treatment options currently available are rarely able to cure metastatic cancer, though some tumors, such as testicular cancer and thyroid cancer, are usually still curable.
- kits for performing the diagnostic and prognostic methods of the invention can be prepared from readily available materials and reagents.
- such kits can comprise any one or more of the following materials: enzymes, reaction tubes, buffers, detergent, primers and probes.
- these kits allow a practitioner to obtain samples of neoplastic cells in blood, tears, semen, saliva, urine, tissue, serum, stool, sputum, cerebrospinal fluid and supernatant from cell lysate.
- these kits include the needed apparatus for performing RNA extraction, RT-PCR, and gel electrophoresis. Instructions for performing the assays can also be included in the kits.
- kits may comprise a plurality of agents for assessing the differential expression of a plurality of biomarkers, wherein the kit is housed in a container.
- the kits may further comprise instructions for using the kit for assessing expression, means for converting the expression data into expression values and/or means for analyzing the expression values to generate scores that predict survival or prognosis.
- the agents in the kit for measuring biomarker expression may comprise a plurality of PCR probes and/or primers for qRT-PCR and/or a plurality of antibody or fragments thereof for assessing expression of the biomarkers.
- the agents in the kit for measuring biomarker expression may comprise an array of polynucleotides complementary to the miRNA biomarkers.
- probes for detecting miRNAs are commercially available. Probes from Applied Biosystems were used for the expression assays that are described in the Examples. The probes of Applied Biosystems may be used to detect the miRNAs discussed herein.
- compositions or components described herein may be comprised in a kit.
- reagents for isolating miRNA, labeling miRNA, and/or evaluating a miRNA population using an array, nucleic acid amplification, and/or hybridization can be included in a kit, as well reagents for preparation of samples from colon samples.
- the kit may further include reagents for creating or synthesizing miRNA probes.
- the kits will thus comprise, in suitable container means, an enzyme for labeling the miRNA by incorporating labeled nucleotide or unlabeled nucleotides that are subsequently labeled.
- the kit can include amplification reagents.
- the kit may include various supports, such as glass, nylon, polymeric beads, magnetic beads, and the like, and/or reagents for coupling any probes and/or target nucleic acids. It may also include one or more buffers, such as reaction buffer, labeling buffer, washing buffer, or a hybridization buffer, compounds for preparing the miRNA probes, and components for isolating miRNA. Other kits of the invention may include components for making a nucleic acid array comprising miRNA, and thus, may include, for example, a solid support.
- Kits for implementing methods described herein are specifically contemplated.
- kits for preparing miRNA for multi-labeling and kits for preparing miRNA probes and/or miRNA anays.
- kit comprise, in suitable container means, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more of the following: (1) poly(A) polymerase; (2) unmodified nucleotides (G, A, T, C, and/or U); (3) a modified nucleotide (labeled or unlabeled); (4) poly(A) polymerase buffer; and, (5) at least one microfilter; (6) label that can be attached to a nucleotide; (7) at least one miRNA probe; (8) reaction buffer; (9) a miRNA array or components for making such an array; (10) acetic acid; (11) alcohol; (12) solutions for preparing, isolating, enriching, and purifying miRNAs or miRNA probes or arrays.
- Other reagents include those generally
- kits may include an array containing miRNA probes, as described in the application.
- An array may have probes corresponding to all known miRNAs of an organism or a particular tissue or organ in particular conditions, or to a subset of such probes.
- the subset of probes on arrays of the invention may be or include those identified as relevant to a particular diagnostic, therapeutic, or prognostic application.
- the array may contain one or more probes that is indicative or suggestive of (1) a disease or condition (thyroid cancer), (2) susceptibility or resistance to a particular drug or treatment; (3) susceptibility to toxicity from a drug or substance; (4) the stage of development or severity of a disease or condition (prognosis); and (5) genetic predisposition to a disease or condition.
- a disease or condition thyroid cancer
- susceptibility or resistance to a particular drug or treatment susceptibility to a particular drug or treatment
- susceptibility to toxicity from a drug or substance susceptibility to toxicity from a drug or substance
- (4) the stage of development or severity of a disease or condition (prognosis) and (5) genetic predisposition to a disease or condition.
- nucleic acid molecules that contain or can be used to amplify a sequence that is a variant of, identical to or complementary to all or part of any of the sequences described herein. Any nucleic acid discussed above may be implemented as part of a kit.
- kits may be packaged either in aqueous media or in lyophilized form.
- the container means of the kits will generally include at least one vial, test tube, flask, bottle, syringe or other container means, into which a component may be placed, and preferably, suitably aliquotted. Where there is more than one component in the kit (labeling reagent and label may be packaged together), the kit also will generally contain a second, third or other additional container into which the additional components may be separately placed. However, various combinations of components may be comprised in a vial.
- the kits of the present invention also will typically include a means for containing the nucleic acids, and any other reagent containers in close confinement for commercial sale. Such containers may include injection or blow molded plastic containers into which the desired vials are retained.
- the liquid solution is an aqueous solution, with a sterile aqueous solution being particularly preferred.
- the components of the kit may be provided as dried powder(s).
- the powder can be reconstituted by the addition of a suitable solvent.
- the solvent may also be provided in another container means.
- labeling dyes are provided as a dried power. It is contemplated that 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 400, 500, 600, 700, 800, 900, 1000 ⁇ g or at least or at most those amounts of dried dye are provided in kits of the invention.
- the dye may then be resuspended in any suitable solvent, such as DMSO.
- the container means will generally include at least one vial, test tube, flask, bottle, syringe and/or other container means, into which the nucleic acid formulations are placed, preferably, suitably allocated.
- the kits may also comprise a second container means for containing a sterile, pharmaceutically acceptable buffer and/or other diluent.
- kits may include a means for containing the vials in close confinement for commercial sale, such as, e.g., injection and/or blow-molded plastic containers into which the desired vials are retained.
- kits may also include components that facilitate isolation of the labeled miR A. It may also include components that preserve or maintain the miR A or that protect against its degradation. Such components may be RNase-free or protect against RNases. Such kits generally will comprise, in suitable means, distinct containers for each individual reagent or solution. [00282] A kit may also include instructions for employing the kit components as well the use of any other reagent not included in the kit. Instructions may include variations that can be implemented.
- Kits may also include one or more of the following: Control RNA; nuclease-free water; RNase-free containers, such as 1.5 ml tubes; RNase-free elution tubes; PEG or dextran; ethanol; acetic acid; sodium acetate; ammonium acetate; guanidinium; detergent; nucleic acid size marker; RNase-free tube tips; and RNase or DNase inhibitors.
- kits are embodiments of kits. Such kits, however, are not limited to the particular items identified above and may include any reagent used for the manipulation or characterization of miRNA.
- Tangible computer-readable medium having computer usable program code executable to perform operations related to early detection, diagnosis or prognosis of cancer.
- a processor or processors can be used in performance of the operations driven by the example tangible computer-readable media disclosed herein.
- the processor or processors can perform those operations under hardware control, or under a combination of hardware and software control.
- the processor may be a processor specifically configured to carry out one or more those operations, such as an application specific integrated circuit (ASIC) or a field programmable gate array (FPGA).
- ASIC application specific integrated circuit
- FPGA field programmable gate array
- the use of a processor or processors allows for the processing of information (e.g., data) that is not possible without the aid of a processor or processors, or at least not at the speed achievable with a processor or processors.
- Some embodiments of the performance of such operations may be achieved within a certain amount of time, such as an amount of time less than what it would take to perform the operations without the use of a computer system, processor, or processors, including no more than one hour, no more than 30 minutes, no more than 15 minutes, no more than 10 minutes, no more than one minute, no more than one second, and no more than every time interval in seconds between one second and one hour.
- Some embodiments of the present tangible computer-readable media may be, for example, a CD-ROM, a DVD-ROM, a flash drive, a hard drive, or any other physical storage device.
- Some embodiments of the present methods may include recording a tangible computer- readable medium with computer-readable code that, when executed by a computer, causes the computer to perform any of the operations discussed herein, including those associated with the present tangible computer-readable media. Recording the tangible computer-readable medium may include, for example, burning data onto a CD-ROM or a DVD-ROM, or otherwise populating a physical storage device with the data.
- MicroRNA expression levels were determined by quantitative real-time PCR (qRT-PCR) and the data were normalized relative to miR-16 expression.
- qRT-PCR quantitative real-time PCR
- MS mass spectrometry
- nanopore amperometry nanopore sequencing, single-molecule, real-time (SM-RT) sequencing, endonuclease digestion, microarrays, matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry, and next-generation sequencing
- next-generation sequencing Laird, Peter W., "Principles and Challenges of Genomewide DNA Expression Analysis," Nature Review Genetics, Vol.
- the present example may include the use of digital color-coded barcode technology analysis (e.g., NANOSTRING® technology (such as the nCounter Analysis System, NanoString Technologies, Inc., Seattle, Washington).
- NANOSTRING® protocol includes the following steps: (1) Hybridization: two ⁇ 50 base probes per mRNA that hybridize in solution, a reporter probe that carries the signal, while a capture probe allows the complex to be immobilized for data collection.
- RKO, SW48, Caco-2, HT29, SW480, and SW620 were obtained from the American Type Culture Collection (ATCC, Rockville, MD). Cell lines were treated with 2.5 ⁇ 5-Aza-2'- deoxycytidine (5-aza-dC; Sigma-Aldrich) for 72 hours, and fresh medium containing 5-aza-dC was replaced every 24 hours.
- Tissue specimens A total of 59 formalin- fixed, paraffin-embedded (FFPE) matched corresponding normal cololectal mucosa (NM), primary CRC tissues (PC), and liver metastasis tissues (LM) were enrolled in this study. Written informed consent was obtained from all patients and the study was approved by the institutional review boards of all participating institutions. Careful microdissection was performed in order to enrich for tumor cells.
- FFPE formalin- fixed, paraffin-embedded
- NM normal cololectal mucosa
- PC primary CRC tissues
- LM liver metastasis tissues
- RNA and DNA Isolation of RNA and DNA.
- Total RNA (including miRNAs) from CRC cell lines was extracted using miRNeasy® Mini Kits (Qiagen).
- miRNeasy® Mini Kits Qiagen
- a Total Nucleic Acid Isolation Kit for FFPE tissues was used according to the manufacturer's instructions.
- DNA was extracted from CRC cell lines using a QIAamp® DNA Mini Kit (Qiagen) and from FFPE specimens using a QIAamp® DNA FFPE Tissue Kit (Qiagen).
- miRNA expression analysis Expression of fourteen metastasis-related miRNAs (let-7i, miR-lOb, miR-30b, miR-34a, miR-141, miR-200b, miR-200c, miR-203, miR-221, miR- 320a, miR-373, miR-429, miR-518d, and miR-520c) was analyzed using TaqMan miRNA assays (Applied Biosystems Inc., Foster City, CA). Expression of RNU6B (Applied Biosystems Inc., Foster City, CA) and miR-16 were used as endogenous controls for cell lines and FFPE tissues, respectively.
- CRC Cell line CAC02, HCT116, RKO, SW48, SW480, and SW620.
- Tissue specimens A total of 59 formalin-fixed, paraffin- embedded (FFPE) primary CRC tissues and corresponding liver metastasis tissues were analyzed.
- FFPE paraffin- embedded
- 5-Aza-2'-deoxycytidine (5-aza-dC) treatment CRC cell lines were treated with 2.5 ⁇ 5-aza-dC for 72 hours
- a-amanitin treatment CRC cell lines were treated with 50 ⁇ g/ml a- amanitin, a RNA pol II inhibitor, for 7 hours.
- Expression analysis Expression levels were analyzed by bisulfite pyrosequencing for quantitative expression analysis using PSQ HS 96A pyrosequencing system (Qiagen) on bisulfite modified genomic DNA template.
- microRNAs expression analysis Expression of miR-373 and miR-520c was analyzed using TaqMan miRNA assays.
- FIG. 1 shows a metastasis predictive microRNAs expression colorectal cancer
- CRC metastasis predictive candidate miRNAs expression status in matched corresponding primary CRC (PC) and liver metastasized CRC (LM) human tissues by qRT-PCR.
- Five miRNAs (let7i, miR-lOb, miR-200b, miR-221, and miR-320) were significantly down-regulated in LM compared to PC.
- three miRNAs (miR-141, miR-200c, and miR-203) were significantly up-regulated in LM compared to PC.
- FIG. 2 is an analysis of the expression of the miR-200 family (-200b, -200c, -141 and -429), and miR-203 in serum samples from CRC patients with metastasis (Stage IV) and without metastasis (Stage I) by qRT-PCR.
- the expression of mir-200c and miR-203 were significantly elevated in serum samples from CRC patients with metastasis (Stage IV) compared to patients without metastasis (Stage I).
- FIG. 3 shows the Results - qRT-PCR validation for selected miRNAs in 58 PCs and LMs.
- FIG. 4 shows the results from the microarray validation for selected miRNAs in 84 PCs (Kaplan-Meier survival curves), in which high expression of has-let-7i and has-miR-320a indicated a good prognosis, which a low expression of has-miR-lOb and has-miR-221 indicated a good prognosis.
- miRNA signature can be used to distinguish between primary CRC and liver metastasis. It was found that a subset of miRNAs, including: let-7i, miR- 10b, miR-30b, miR-200b, miR-320a, and miR-518d were significantly downregulated in liver metastasis tissues compared to primary CRC. In contrast, miRNAs such as miR-141, miR-200c, and miR-203 were significantly over-expressed in liver metastasis tissues. In a further evaluation step using serum samples from CRC patients, it was found that the serum expression levels of miR-200c and miR-203 were upregulated in CRC patients with distant metastasis compared to CRC patients without metastasis.
- the screening step included the following materials: 9 pairs of primary CRC
- PC liver metastasis
- LM liver metastasis
- a microarray validation step included the following materials: 84 pairs of PC and corresponding normal mucosa (NM), frozen tissue, not-microdissected.
- the method used was MicroRNA microarray (quadruplicates of 389 human miRNAs) as published in JAMA. 2008 Jan 30;299(4):425-36.
- a qRT-PCR Validation step included the following materials: 175 PCs, FFPE tissue, microdissected.
- the method for analysis was TaqMan miRNA assays, with miR-16 used as endogenous control.
- Table 1 is a summary of the clinicopathology characteristics of the colorectal cancer patients.
- Table 2 shows the 10 miRNAs differentially expressed in matched PCs and LMs using the NANOSTRING® screening step.
- Table 3 shows the results from the microarray validation for 4 miRNAs in 84
- let-7i, miR-lOb and miR-320a m PC were significantly associated with the distant metastasis, while the expression of let-7i and miR-lOb was significantly associated with the TNM stage.
- hsa-let-7i hsa-miR-lOb hsa-miR-221 hsa-miR-320a mean+SD P value mean+SD P value n mean+SD P value mean+SD P value
- Table 4 shows the results of microarray validation for 4 miRNAs in 84 PCs, using a Cox proportional hazards model. It was found that low expression of let-7i was an independent prognostic factor.
- hsa-let-7i (Low vs. High) 2.6706 1.2577 to 5.6710 hsa-miR-320a (Low vs. High) 1.6783 0.8655 to 3.2545 0.1239
- Table 5 shows the results of microarray validation for 4 miRNAs in 84 PCs, using a logistic regression model. It was found that all 4 miRNAs (let-7i, miR-320a, miR-lOb and miR-221) expression in PCs was significantly associated with the distant metastasis. It was also found that low expression of let-7i and high expression of miR-lOb in PCs were an independent metastasis prediction marker, respectively.
- Figure 5 shows the results of qRT-PCR validation for miR-7i and miR-lOb in 175
- Table 6 shows the results of qRT-PCR validation for miR-7i and miR-lOb in 175
- Table 7 shows the results from qRT-PCR validation for miR-7i and miR-lOb in
- CEA >Median vs. ⁇ Median
- CEA >Median vs. ⁇ Median
- Table 8 shows the results from qRT-PCR validation for miR-7i and miR-lOb in
- Figure 6 shows the ISH validation for the expression of miR-7i and miR-lOb in
- Example 2 Identification of a metastasis-specific miRNA signature in human colorectal cancer
- a metastasis-specific miRNA biomarker discovery approach was developed that involved comprehensive miRNA expression profiling of both primary CRC (pCRC) and matched liver metastasis (LM) tissues, followed by validation of these newly discovered miRNA biomarkers in multiple, large, independent cohorts of tissues.
- the expression patterns of these miRNAs was correlated with important clinicopathological parameters related to CRC metastasis.
- the feasibility of these miRNAs as tissue-based and serum-based metastasis-specific biomarkers was also evaluated.
- Adenocarcinoma includes well, moderately, and poorly differentiated.
- Nanostring assay and miRNA microarray analysis 100 ng of total RNA from frozen tissues were analyzed using NanoString human miRNA vl assays (NanoString Technologies, Seattle, USA) according to the manufacturer's instructions (NCBIGEO GSE44121). After background subtraction, data was normalized to the geometric mean of the top 75 miRNAs. Paired Student's t-test was used to calculate statistical significance.
- the miRNA microarray expression data (NCBIGEO GSE7828) (Schetter 2008) were analyzed for miRNA expression in primary tumors that were associated with distant metastases.
- RNA was extracted from FFPE specimens using the Total Nucleic Acid Isolation Kit for FFPE tissues (Ambion, Austin, TX, USA). RNA was extracted from frozen tissue specimens using standard Trizol protocols (Invitrogen). Expression of let-7i, miR-lOb, miR-30b, miR-221, miR-320a, and miR-885-5p were analyzed using TaqMan miRNA assays (Applied Biosystems, Foster City, CA), and miR-16 expression was used as an endogenous control for data normalization, as previously described (Hur 2012).
- RNAs were isolated from 250 > ⁇ serum samples from CRC patients using the Qiagen miRNAeasy Kit (Qiagen, Valencia, CA). For normalization of sample-to-sample variation during RNA isolation, 25 fmol of synthetic C. elegans miRNA (cel-miR-39, Applied Biosystems) was added to each serum sample. Expression of miR-885-5p was analyzed using TaqMan miRNA assays (Applied Biosystems) as described previously (Hur 2012).
- ISH In situ Hybridization
- Positive controls U6 snRNA, LNA-modified and 5'- and 3'- DIG-labeled oligonucleotide, Exiqon
- negative controls scrambled miRNA control, LNA- modified and 5'- and 3'-DIG-labeled oligonucleotide, Exiqon
- Statistical Analysis Paired t-test, Mann- Whitney test, and ⁇ 2 tests were used to analyze microRNA expression. Kaplan-Meier analysis and the log-rank test were used for survival analysis.
- ROC receiver operating characteristic
- Univariate and multivariate Cox's propmiional hazards models were used to identify independent prognostic factors dictating patient survival.
- Univariate and multivariate logistic regression models identified independent predictive factors for distant metastasis and lymph node (LN) metastasis. Data are presented as mean ⁇ S.D. (standard deviation) and all statistical analyses were conducted using the Medcalc version 12.3 (Broekstraat, Belgium) and the GraphPad Prism version 5.0 (GraphPad Software, San Diego, CA).
- LM tissues To discover miRNAs that are involved in CRC metastasis, NanoString-based miRNA expression profiling was performed in 9 pairs of matched pCRC and LM frozen tissues (Table 10). Twenty three miRNAs were found to be differentially expressed between pCRC and matched LM tissues (p ⁇ 0.001). All but 3 miRNAs (miR-122, miR-30b, and miR-885-5p) were down-regulated in LM. Since miR-122 is a conserved liver-specific miRNA and is required for normal hepatic biology and function (Chang 2004), this miRNA was excluded from further analysis.
- miRNAs 10b, and miR-221) and 2 up-regulated miRNAs were selected for validation by qRT-PCR in an independent cohort of 59 matched pCRC and LM tissues (Fig. 7). All but one miRNA was validated, including all of the down-regulated miRNAs (let-7i, 0.62
- T stage (T3/4 vs. Tl/2) 0.7021 to 0.5217 to
- T stage (T3/4 vs. Tl/2) 0.7021 to 0.5675 to
- hsa miR-221 (Low vs. High) 0.9687 to 0.7260 to
- T stage (T3/4 vs. Tl/2) 0.0000 to 0.0000 to
- hsa-miR-320a (Low vs. High) 1.3048 to 0.1682 to
- T stage (T3/4 vs. Tl/2) 0.0000 to 0.0000 to
- Sex (Male vs. Female) i isn,. 0.6133 tocroft , and, . 0.8779 to ⁇ 1 1 1 1 0
- T stage (T3/4 v, Tl/2) ⁇ 1.9318 to ⁇ ⁇ 0.3924 to ⁇
- T stage (T3/4 vs. Tl/2) 1.8281 to 0.4865 to
- TNM stage 0.0235
- liver metastasis 0.0011
- lymphatic invasion 0.141
- Tissue expression of miR-885-5p expression was not associated with clinico-pathological factors.
- Tumor size (>40mm vs. >40mm) 1.2735 to 0.2918 to
- Serum miR-885-5p (High vs. 1.9139 to 1.0985 to
- Tumor size (>40mm vs. ⁇ 40mm) 1.2759 to 0.8232 to
- Serum miR-885-5p (High vs. 1.1082 to 1.2821 to
- Tumor size (>40mm vs. ⁇ 40mm) 1.5934 to 0.5150 to
- Lymph node metastasis (Present 9.7845 to 4.3232 to
- Serum miR-885-5p (High vs. 1.4481 to 0.9731 to
- ISH in situ hybridization
- ISH analysis showed low let-7i and high miR-lOb expression in late stage pCRCs with metastasis compared to early stage pCRCs without metastasis, while miR-885-5p expression was comparable between early stage pCRCs without metastasis and late stage pCRCs with metastasis.
- Down-regulated expression of let-7i and miR-lOb and up-regulated miR-885-5p expression was observed in LM compared to pCRC.
- adjacent hepatocytes barely expressed any of the 3 CRC metastasis-specific miRNAs.
- Example 3 - Serum miR-21 as a promising biomarkers for the early detection and prognosis of colorectal cancer
- Study Design The present study included analysis of 568 serum and tissue specimens that were obtained from healthy volunteers and consecutively enrolled patients with colorectal adenomas and cancers, at the Mie University Medical Hospital, Japan, between January 1 2005 and December 31, 2010. This study was designed as an initial screening phase and a subsequent validation phase. In the screening phase, oncogenic miR-21 and miR-31 were selected (Meng 2007; Zhu 2007; Asangani 2008; Wang 2009; Cottonham 2008), and their expression was measured usmg TaqMan-based quantitative RT-PCR (qRT-PCR) using cell culture medium and matched serum and tissue samples.
- qRT-PCR quantitative RT-PCR
- CRC cell lines HCT 116 and SW620
- a fraction of culture medium was collected at 0, 24 and 48h after the initial seeding of cells in 10 cm dishes.
- pre-operative serum samples were collected from 12 CRC patients and from 12 gender- and age-matched healthy subjects as controls.
- CRC and adjacent normal tissues were analyzed from 8 of the 12 CRC patients, from whom both matched normal and neoplastic tissues were available.
- RNA isolation and qRT-PCR MiRNA extraction from serum and culture media samples was performed with miRNeasy RNA isolation Kit (Qiagen, Valencia, CA), while those from FFPE samples using RecoverAll Total Nucleic Acid Isolation Kit (Ambion Inc., Austin, TX). TaqMan miRNA real-time RT-PCRs (Applied Biosystems, Foster City, CA) were employed to detect and quantify the miRNAs expression using the 2 " method.
- Statistical analyses Results were expressed as mean +SD (standard deviation). Mann-Whitney U and Kruskal-Wallis tests of variance (ANOVA) were used to evaluate statistical differences in serum or tissue miRNA expression between unpaired groups, and multiple comparison groups, respectively. Wilcoxon test was used to compare miR-21 expression in paired serum samples obtained pre- and 7 day post-surgical tumor resection. The Spearman's correlation test was used to examine correlation between miRNA expression inserum and matched CRC tissues. Receiver operating characteristic (ROC) analysis was performed to determine the diagnostic performance of miR-21 expression levels in distinguishing patients with colorectal adenomas or cancers from the healthy controls.
- ROC Receiver operating characteristic
- Sensitivity against 1 -specificity was plotted at each cut-off threshold, and the area under the curve (AUC) values that reflect the probability of correctly identifying adenoma or CRC patients from controls were computed.
- the optimal cut-off threshold for diagnosis were obtained by Youden Index (Ruopp 2008). In brief, the optimal cut-off threshold values were determined at the point on ROC curve at which Youden' s Index (sensitivity+specificity-1) was maximal. By using these optimal cut-off values, sensitivity, specificity, Positive and Negative Predictive Values (PPV and NPV) were calculated.
- BCa bootstrap bias-correction and accelerated (BCa) bootstrap methods were performed (Efron 1987). In general, there are no standard recommended methods for adjusting bias. However, BCa bootstrap method was selected because it adjusts for both bias and skewness in the bootstrap distribution of data. For this analysis, data were randomly included from the original serum samples, followed by sensitivity and 1 -specificity determination for various cut-off thresholds. This process was repeated 1000 times, and resultant mean values (95% confidence interval) for sensitivity and specificity were computed.
- ROC convex hull (ROCCH) curves were generated using approaches that allowed hull segment to be viewed as being generated by a tie between the plots of sensitivity and specificity for various cutoffs from the original data and adjusted data by BCa bootstrap methods. Thereafter, the AUCs of ROCCH curves were calculated by Trapezoidal rule. Finally, a two-sided z-test was used to compare the AUCs of two ROC curves (Hanley 1983). Multivariate logistic regression model was used to calculate odds ratios (ORs) for age and gender adjusted cases being associated with CRC or adenoma according to serum miRNA levels.
- Cox's proportional hazard regression analyses were used to estimate hazard ratios of death according to serum and tumor miRNA levels, unadjusted and adjusted for potential confounding factors for death including age, gender, pathological differentiation, T stage, N stage, M stage and serum CEA levels. All -values are two-sided; ⁇ 0.05 was considered significant. All statistical analyses were carried out using Medcalc vl2.3.0 (Broekstraat 52, 9030, Mariakerke, Belgium).
- MiR-21 expression in colorectal cancer cell culture medium In this initial experiment, it was determined whether miR-21 and miR-31 act as secretory miRNAs and are excreted into the culture media by HCT116 and SW620 CRC cell lines. miR-21 expression in the culture media from both cell lines increased with time (24 and 48 hours; ⁇ 0.05), and with increasing numbers of tumor cells ( ⁇ 0.05); (Figure 1A:HCT116, Figure IB: SW620). However, miR-31 expression levels did not show significant changes in either cell line (Figure 1C: HCT116, Figure ID: SW620), suggesting that miR-21 but not miR-31, is a secretory miRNA.
- miR- 21 levels were significantly elevated in the sera of CRC patients ( ⁇ 0.0001; Figure 13G), while no significant differences were noted in serum miR-31 expression between CRC patients and controls ( >0.05; Figure 13H). Based on the results that only miR-21 acts a secretory miRNA the next study focused on miR-21 for further assessment of its efficacy as a diagnostic and prognostic biomarker in patients with colorectal neoplasia.
- Tissue and serum miR-21 expression during validation phase Patient characteristics— The clinicopathological and other patient characteristics are summarized in
- the median follow-up time period for CRC patients was 44 months (range: 2-84 months).
- Table 17 Patient characteristics for serum and tissue miR-21 expression analysis in the validation set
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-
2014
- 2014-03-17 WO PCT/US2014/030407 patent/WO2014145612A1/fr active Application Filing
- 2014-03-17 US US14/215,959 patent/US9868992B2/en active Active
- 2014-03-17 EP EP14764952.9A patent/EP2971132B1/fr active Active
- 2014-03-17 CA CA2907377A patent/CA2907377A1/fr not_active Abandoned
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WO2014145612A1 (fr) | 2014-09-18 |
US20140322354A1 (en) | 2014-10-30 |
CA2907377A1 (fr) | 2014-09-18 |
EP2971132A4 (fr) | 2016-10-19 |
EP2971132B1 (fr) | 2020-05-06 |
US9868992B2 (en) | 2018-01-16 |
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